RU2810003C2 - mRNA PURIFICATION BY TANGENTIAL FLOW FILTRATION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: a method of purifying mRNA molecules and a method of producing a pharmaceutical composition including the step of purifying mRNA using the above method of purifying mRNA molecules. The purification method includes the steps of (Ia) purifying precipitated mRNA molecules from a suspension including precipitated mRNA molecules using a first solution, (Ib) washing and dissolving the purified precipitated mRNA molecules obtained in step (Ia) using a second solution, (IIa) purifying the mRNA molecules from the dissolved mRNA molecules obtained in step (Ib) using a third solution including a chelating agent, and (IIb) washing the purified mRNA molecules obtained in step (IIa) using a fourth solution. Stages (Ia)-(IIb) are carried out using tangential flow filtration.

EFFECT: invention expands the arsenal of high-throughput methods of obtaining purified mRNA molecules.

15 cl, 12 dwg, 13 tbl, 1 ex

Description

The present invention relates to a method for purifying mRNA molecules, comprising (Ia) purifying precipitated mRNA molecules from a suspension comprising precipitated mRNA molecules, (Ib) washing and dissolving the purified precipitated mRNA molecules, (IIa) purifying the mRNA molecules using a solution including a chelating agent , followed by (IIb) washing of the purified mRNA molecules, where stages (Ia)-(IIb) are carried out using tangential flow filtration.

Genetic information is stored in the cell as deoxyribonucleic acid (DNA) and can be transcribed into ribonucleic acid (RNA) when needed. Both DNA and RNA molecules are composed of nucleotides consisting of a nitrogenous base, a five-carbon sugar, and at least one phosphate group. There are different types of RNA molecules, including mRNA molecules, which carry the genetic information for protein synthesis. In eukaryotes, these mRNA molecules are transcribed from DNA as immature mRNA molecules and are subsequently modified by the addition of, for example, a 5' cap and a 3' polyadenosine (poly(A)) tail. Mature mRNA molecules are then transferred from the cell nucleus to the cytoplasm, where they are translated into proteins. Thus, the DNA sequence, as well as the quantity, stability and translational efficiency of mature mRNA molecules, mainly determine the synthesis of proteins in the cell.

To optimize the synthesis of a desired protein in a cell, for example for therapeutic purposes, mRNA-based approaches represent a promising tool. The advantage of using mRNA molecules is that the molecules only need to be introduced into the cytoplasm of the cell for protein translation (Tavernier et al., 2011, J Control Release, 150 (3): 238-247, Yamamoto et al., 2009, Eur J Pharm Biopharm, 71 (3): 484-489). In addition, the use of mRNA molecules is a simpler and more efficient method than using the corresponding DNA sequence on a suitable carrier such as a plasmid, and eliminates the significant risk of alteration of chromosomal DNA if the plasmid or parts thereof are incorporated into the genome. mRNA molecules are mainly produced by in vitro transcription, for example for therapeutic purposes. Such approaches are based on template DNAs, which are generated either by linearization of the plasmid DNA itself or by polymerase chain reaction (PCR) from the plasmid DNA. Both approaches are well known and can be scaled up using simple methods. However, in both cases, the mRNA molecules must be purified before they can be used in the next steps of their application.

Purification of synthesized mRNA molecules is critical because contaminants negatively affect most downstream applications, primarily for therapeutic purposes. Various by-products can contaminate the desired mRNA molecules, such as components used and/or generated in previous steps such as mRNA synthesis. In the case of both approaches, in vivo and in vitro, short fragments of the mRNA molecule can be formed, for example, as a result of hydrolysis and interrupted transcription. Hydrolysis of an mRNA molecule can occur in the presence of a catalyst or enzyme, or spontaneously, leading to degradation of the corresponding mRNA molecule and, thus, to the formation of fragments of the mRNA molecule of variable length. In the case of interrupted transcription (see, for example, Revyakin et al., 2006, Science, 314 (5802): 1139-1143), RNA polymerase binds to the appropriate promoter element in the DNA molecule for transcription, thereby unwinding the double strand of DNA, surrounding the transcription start site and the synthesis of the mRNA molecule begins. However, in some cases, RNA polymerase does not separate from the promoter region of the DNA, but remains stationary while transcribing part of the DNA molecule. As the unwound DNA accumulates within the enzyme, RNA polymerase pushes out the portion of the DNA that stores genetic information for transcription and releases a synthesized RNA molecule ranging from one to 15 nucleotides in length (see, for example, Goldman et al., 2009, Science , 324 (5929): 927-928). To avoid, for example, an unwanted immune response after administration of a pharmaceutical composition comprising such short fragments of mRNA, there is an urgent need to obtain purified unfragmented mRNA molecules for their further use, especially for therapeutic purposes.

There are various approaches to purify mRNA molecules, including precipitation and filtration, as well as combinations thereof. For the removal of unwanted salts, nucleotides and proteins, RNA precipitation approaches provide, for example, a simple and cost-effective way to obtain purified RNA molecules in the form of a pellet that can be resuspended in the desired buffer solution. Various solutions can be used for precipitation, containing, for example, guanidine thiocyanate (GSCN), ammonium acetate (NH ₄ OAc), ethanol, lithium chloride and sodium acetate (for a review, see, for example, Walker and Lorsch, 2013, Methods Enzymol, 530: 337-343). On the other hand, filtration methods rely on a mechanical concept to purify target molecules such as mRNA molecules. In the case of filtration, the feedstock, such as a solution or suspension, including target molecules such as mRNA, is passed through or over a filtration membrane, and the latter method is called tangential flow filtration (TFF).

WO 2014/152966 A1 and WO 2015/164773 A1, for example, describe methods for purifying mRNA molecules using TFF and various volumes of solution and/or suspension. The mRNA molecules are purified by precipitating the mRNA molecules using, for example, urea, GSCN and/or potassium chloride, and the precipitates are washed several times. However, in both cases it was not found that this could purify the target mRNA molecules, taking into account short fragments of mRNA, such as immature mRNA molecules and/or hydrolysis products.

Application WO 2016/193206 A1 describes a method for the production and purification of RNA, where the purification of the RNA includes at least one TFF step and preferably does not include a step involving phenol/chloroform extraction and/or DNA and/or RNA precipitation. Moreover, the RNA molecules are preferably purified using an additional purification step using, for example, cation and/or anion exchange chromatography, and/or reverse phase chromatography. As stated above, the document does not disclose the purification of target RNA molecules taking into account immature mRNA molecules, and/or hydrolysis products of mRNA molecules.

Application WO 2018/157133 A1 describes methods for purifying mRNA molecules on a preparative scale. The invention relates primarily to the use of a stirred cell or a shaken suction filter to obtain transparent and homogeneous mRNA compositions. As described in the Examples section, mRNA molecules are precipitated using GSCN and the precipitate is washed using a solution containing GSCN and ethanol. It should be noted that the compositions of mRNA molecules obtained by the claimed method are compared with compositions including mRNA molecules purified using TFF, and it was found that only TFF-based purification leads to a solution containing residual enzymes in addition to the target mRNA molecules.

Therefore, there is still a need to have alternative solutions on hand to efficiently purify mRNA molecules on a preparative scale, especially taking into account immature mRNA molecules and hydrolysis products of mRNA molecules, ensuring low levels of salts that may pose problems in downstream applications, such as GSCN, and with a system that can be automated in a simple way.

The present application addresses the need to obtain purified mRNA molecules with high throughput when implementing the embodiments set forth in the claims.

First of all, the present invention relates to a method for purifying mRNA molecules, the method comprising (Ia) purifying precipitated mRNA molecules from a suspension comprising precipitated mRNA molecules using a first solution, (Ib) washing and dissolving the purified precipitated mRNA molecules obtained in step ( Ia), using a second solution, (IIa) purifying the mRNA molecules from the dissolved mRNA molecules obtained in step (Ib), using a third solution including a chelating agent, followed by (IIb) washing the purified mRNA molecules obtained in step (IIa) ) using a fourth solution, where steps (Ia)-(IIb) are carried out using tangential flow filtration.

Surprisingly, it was found that the method of the present invention not only leads to a high degree of purification of the mRNA molecules and, thus, to obtaining a solution containing purified mRNA molecules, but also containing no or virtually no hydrolysis products of mRNA molecules and immature mRNA molecules. First of all, it was found that when using this method, hydrolysis products of mRNA molecules, immature mRNA molecules, proteins and salts, which can create problems at subsequent stages of application, are effectively removed. Thus, using the method of the present invention, it is possible to obtain a solution comprising purified mRNA molecules, suitable for subsequent use in areas such as pharmaceuticals.

In the context of the present invention, the term "purification method" refers to a method of obtaining target molecules from a mixture, for example, a solution or suspension, including the specified target molecules intended for purification and other components other than the target molecules, where the concentration of the target molecules is increased or increased in solution obtained after implementation of the specified method compared to the concentration of the target molecules in the mixture before implementation of the specified method. Purification of target molecules can also be referred to as enrichment of said target molecules by removing, or at least substantially removing, components other than the target molecules.

In the context of the present invention, the target molecules are mRNA molecules. As used herein, the term "mRNA" and the term "mRNA molecule", which are used interchangeably, refer to a single-stranded RNA molecule constructed, for example, from nucleotides A, C, G and/or U, that is, nucleotides including adenine, guanine, cytosine and uracil, as the corresponding nitrogenous base. Moreover, the mRNA molecule contains one or more coding sequences that can be used as a template during the synthesis of the amino acid sequence during translation. Thus, the mRNA molecule to be purified preferably includes a coding sequence, that is, a sequence that can be translated into an amino acid sequence such as a protein, and includes a start codon and a stop codon. The coding sequence may be a natural sequence, a partially or completely codon-optimized sequence derived from the natural sequence to be used, or an artificial sequence. Codon optimization refers to a technique that is used to maximize protein expression by increasing the translation efficiency of the corresponding mRNA molecule, since in some cases there are codons that are preferentially used by certain species for a given amino acid. Moreover, said mRNA molecule may contain a 5' and/or 3' untranslated region (UTR), one or more internal ribosome entry sites (IRES), one or more additional sequences to stimulate translation, and/or one or more additional modifications to regulate and/or increasing the duration of action. Other features of mRNA molecules are described in more detail below.

Thus, the term “mRNA” is understood to mean any RNA molecule that is suitable for expressing an amino acid sequence or that is translated into an amino acid sequence, such as a protein. Thus, the mRNA molecule is assumed to exhibit the property and/or activity by which it is characterized, and thus can be translated into an amino acid sequence, such as a protein with the specified amino acid sequence exhibiting the activity and/or property by which it is characterized.

As used herein, the term "amino acid sequence" covers any type of amino acid sequence, that is, chains of two or more amino acids that are linked to each other through peptide bonds, and refers to any amino acid sequence of interest. Preferably, the length of the encoded amino acid sequence is at least 5 amino acids, more preferably at least 10 amino acids, even more preferably at least 50, 100, 200 or 500 amino acids. Thus, the term "amino acid sequence" covers short peptides, oligopeptides, polypeptides, fusion proteins, proteins, as well as fragments thereof, such as fragments of known proteins, preferably functional fragments. For example, such fragments may be biologically active protein fragments or antigenic fragments, such as epitopes, which may be effective in increasing antibody levels. As for the function of the amino acid sequence, there is no limitation, and possible amino acid sequences are described in detail below.

In contrast, components to be removed during purification of said mRNA molecules, and thus components other than the target molecules, include, for example, mRNA molecules that are partially or completely degraded by hydrolysis. Degradation by hydrolysis is a random process and the resulting degradation products, in this context called “hydrolysis products” or also “mRNA molecule hydrolysis products,” are shortened by at least 1 nucleotide compared to the mRNA molecule that was hydrolyzed. Thus, depending on the length of the mRNA molecule that has been hydrolyzed, the length of the resulting hydrolysis products may be, for example, less than 10,000 nucleotides, preferably less than 5,000 nucleotides, more preferably less than 500 nucleotides, even more preferably less than 250 nucleotides, even more preferably less than 170 nucleotides. nucleotides and most preferably less than 120 nucleotides. Another example of components to be removed are immature mRNA molecules that are, for example, less than 50 nucleotides in length, preferably less than 25 nucleotides, and more preferably less than 15 nucleotides.

Moreover, components intended to be removed during purification of mRNA molecules include, for example, components that were used during in vitro synthesis of target molecules, such as in vitro transcription of mRNA molecules and/or components included in cells used for template amplification for the synthesis of target molecules and/or for the transcription of mRNA molecules. In the case of in vitro transcription of mRNA molecules, such components include, for example, proteins such as enzymes used for in vitro transcription, salts, ions such as magnesium ions, nucleoside triphosphates, mono-, di- and/or triphosphates including 5' cap and/or 5' cap analogues, immature transcripts, hydrolysis products, a buffer such as 3-(N-morpholino)propanesulfonic acid (MOPS) and/or a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

The method of the present invention is carried out by tangential flow filtration (TFF) method. The TFF method is characterized in that a suspension or solution containing the mRNA molecules to be purified flows in a tangential flow over the surface of the filtration membrane, with the mRNA molecules preferentially retained in concentrate. Therefore, by performing TFF to purify mRNA molecules, the formation of filter cakes that can clog the filtration membrane over time is avoided, and thus the useful life of the filtration membrane can be extended.

Thus, the target molecule purification system of the present invention is a TFF system. A TFF system typically includes a filtration device such as a capsule, cassette and hollow fiber holder or module, a pump, hose, valve or clamp and fluid reservoir, and optionally a pressure gauge (see FIG. 1). The TFF system can be used continuously even at high concentrations of mRNA molecules without clogging the pores of the filtration membrane. The use of the TFF system is advantageous because it allows high throughput purification of mRNA molecules.

It should be noted that the term "solution" usually refers to a homogeneous mixture of two or more substances and is primarily applied to the liquid state of a substance, although solutions of gases and solids are also possible. In this context, the term "solution", especially in the case of the "first solution", "second solution", "third solution" and "fourth solution" in accordance with the present invention includes a liquid such as water, preferably a homogeneous mixture of liquids, as well as , for example, a mixture of liquid and preferably dissolved salt.

The present invention provides a method for purifying mRNA molecules, wherein the method comprises the step (Ia) of purifying precipitated mRNA molecules from a suspension that includes said precipitated mRNA molecules. This cleaning is achieved using the first solution. A suspension comprising precipitated mRNA molecules can be obtained, for example, under conditions of cell lysis or in vitro transcription. Thus, the precipitated mRNA molecules should be purified from cellular components or from in vitro transcription mixtures, such as amino acid sequences including proteins and enzymes, nucleoside triphosphates, components of buffer solutions and optionally 5' cap and/or 5' cap analogues. 5' Cap and/or 5' cap analogues may be present in the suspension used for step (Ia), for example, in the case where the mRNA molecules have been transcribed in vitro and co-transcriptionally 5' capped. Therefore, it is advantageous to step (Ia) to remove cellular components or components from in vitro transcription mixtures from precipitated mRNA molecules.

In some embodiments of the present invention, the first solution contains ammonium acetate (NH ₄ OAc). Thus, the precipitated mRNA molecules are purified from the suspension using a first solution containing NH ₄ OAc. Moreover, the pH value of the first solution is preferably in the range from 1 to 12, more preferably from 1 to 10, even more preferably in the range from 3 to 9, and most preferably in the range from 6 to 8. Thus, the precipitated mRNA molecules are purified from a suspension comprising precipitated mRNA molecules, using a first solution comprising NH ₄ OAc and having a pH value in the range of 1 to 12, preferably in the range of 1 to 10, more preferably in the range of 3 to 9, and even more preferably in the range from 6 to 8.

The method for purifying mRNA molecules of the present invention further includes the step (Ib) of washing and dissolving the purified precipitated mRNA molecules obtained in step (Ia) described above using a second solution. Said second solution may be any solution in which the mRNA molecules can be resuspended and in which the mRNA molecules remain stable, that is, not hydrolyzed, such as water. Carrying out step (Ib) is advantageous for removing certain components of the first solution and for obtaining dissolved mRNA molecules. In some embodiments of the present invention, the second solution is water.

The method for purifying mRNA molecules of the present invention further includes the step (IIa) of purifying the mRNA molecules from the dissolved mRNA molecules obtained in step (Ib) using a third solution including a chelating agent. This step is advantageous for removing immature mRNA molecules and hydrolysis products from a solution containing dissolved mRNA molecules.

The term "chelating agent" means molecules that chelate cations and especially molecules with donor groups that can bind to the central atom in the coordination complex depending on several factors, such as pH and/or temperature, the concentration of the chelating agent and/or the type of chelating agent cation. Chelating agents can be characterized, for example, by the number of "dentates", that is, the number of donor groups per molecule and/or the number of coordinating positions on the chelating cation. For example, the chelating agent EDTA contains six “dentates” and chelates the divalent cation in a 1:1 complex of the divalent cation and the chelating agent.

Preferably, in the context of the present invention, the term "chelating agent" refers to chelating agents that are capable of complexing divalent and/or trivalent cations, such as, for example, divalent magnesium or calcium ions and/or ferric ions. Generally, chelating agents containing less than four cationic coordination positions have lower chelating activity compared to chelating agents including four or more cationic coordination positions, primarily in view of the efficient complexation of divalent and/or trivalent cations.

The chelating agents of the present invention are chelating agents comprising at least four coordination positions on the chelating cation, more preferably chelating agents comprising more than four coordination positions on the chelating cation, even more preferably at least four dentates per molecule, in this context also are called "highly effective chelating agents", more preferably chelating agents comprising more than four coordinating positions on the chelating cation, which are used in pharmaceutical compositions and/or formulations, and even more preferably EDTA. Chelating agents comprising more than four coordinating positions on the cation being chelated, which are used in pharmaceutical compositions, for example, are used in antidotes containing a solution of Na ₂ EDTA (for example, 2 g/10 ml, see, for example, GPUPharma GmbH) and/or disodium -calcium salt EDTA (for example, 50 mg/ml, 10 ml ampoule, see, for example, Laboratories SERB), or, for example, used as excipients (see, for example, Phenhydan, Desitin Arzneimittel GmbH, or Soluvit N, Baxter , or Fluimucil, Zambon GmbH).

Thus, in some embodiments of the present invention, the chelating agent is EDTA.

Typical preferred chelating agents are listed in Table. 1, with EDTA being the most preferred chelating agent.

Highly effective chelating agents such as EDTA can effectively remove primarily divalent cations such as magnesium cations. Magnesium cations, for example, are required for enzymes that take part in the transcription of mRNA molecules and are thus present in large quantities in cell mixtures and in vitro transcription mixtures. The inventors of the present invention have surprisingly found that effective removal of hydrolysis products and immature mRNA molecules is achieved by using a highly effective chelating agent such as EDTA in step (IIa), while providing a high degree of purification of the mRNA molecules. Without being bound by any theory, it can be speculated that the above cations may contribute to the formation of secondary and tertiary structures of mRNA molecules, and that the removal of primarily divalent cations, such as magnesium cations, may be of particular importance for the purification of mRNA molecules, since the molecules mRNA as immature mRNA molecules and hydrolysis products may tend to bind to purified mRNA molecules in the presence of these cations. Thus, it can be assumed that by removing divalent cations using a highly effective chelating agent such as EDTA in step (IIa), the melting temperature is reduced and the secondary and/or tertiary structure of the mRNA molecules is affected.

Thus, immature mRNA molecules and hydrolysis products can be removed as a filtrate. Therefore, step (IIa) is primarily advantageous for removing immature mRNA molecules and hydrolysis products.

According to the present invention, the third solution includes a chelating agent, preferably a high performance chelating agent as defined above, most preferably EDTA. As stated above, the complexing ability of chelating agents can be influenced by the pH value, since the complexing constant of a given chelating agent is influenced by the pH value. For example, EDTA is most effective at higher pH values, such as 8 to 10, at chelating magnesium ions. However, it is desirable to adjust the pH value taking into account the optimal compromise between the complexing ability of a given chelating agent and the stability of the mRNA molecules being purified. In this context, it has been found that a highly effective chelating agent such as EDTA can effectively remove primarily divalent cations at a pH of 1 to 10, preferably 6 to 8. Thus, without being bound by any theory, it is believed that This melting temperature, especially of immature mRNA molecules and hydrolysis products that are associated with mRNA molecules, can be lowered using highly effective chelating agents such as EDTA, and thus immature mRNA molecules and hydrolysis products can be removed while simultaneously adjusting the pH to a value which maintains the stability of the purified mRNA molecules.

Thus, in a preferred embodiment of the present invention, the pH value of the third solution containing the chelating agent is adjusted to a pH value at which the corresponding chelating agent has optimal chelating ability.

Therefore, the use of, for example, a highly effective chelating agent such as EDTA, at a pH in the range of 1 to 10, preferably 6 to 8, is advantageous for the effective removal of divalent cations, and thus, in turn, for the effective removal of immature molecules mRNA and hydrolysis products. If one of the alternative chelating agents mentioned above is used, it may be necessary to adjust the pH to a value at which the alternative is effective in removing ions, preferably divalent magnesium cations. The appropriate pH ranges for preferred chelating agents are listed in the table above.

In some embodiments, the pH of the third solution is in the range of 1 to 10, preferably in the range of 3 to 9, and more preferably in the range of 6 to 8.

The pH value of the third solution can be adjusted using a buffer substance. Preferred buffering agents (their respective pH ranges are indicated in parentheses) include 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-Tris, pH range 5.8-7, 2), N-(2-acetamido)iminodiacetic acid (ADA, pH range 6.0-7.2), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES, pH range 6.1-7.5 ), 1,4-piperazinediethanesulfonic acid (PIPES, pH range 6.1-7.5), 2-hydroxy-3-morpholin-4-ylpropan-1-sulfonic acid (MOPSO, pH range 6.2-7.6 ), 1,3-bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris propane, pH range 6.3-9.5), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES, pH range 6.4-7.8), 3-(N-morpholino)propanesulfonic acid (MOPS, pH range 6.5-7.9), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane -2-yl]amino]ethanesulfonic acid (TES, pH range 6.8-8.2), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH range 6.8- 8.2), 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO, pH range 7.0-8.2), 4-(N-morpholino)butanesulfonic acid (MOBS , pH range 6.9-8.3) and 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO, pH range 7 ,0-8.2). A particularly preferred buffering agent is MOPS, having a preferred pH of 6 to 8, more preferably 6.5 to 7.5.

In some embodiments, the pH of the third solution is in the range of 1 to 10, preferably 3 to 9, more preferably 6 to 9, and said solution includes a buffering agent, preferably MOPS.

In some embodiments, the third solution includes a MOPS buffer and is preferably prepared by adjusting the pH with the MOPS buffer before adding a chelating agent, preferably EDTA.

Thus, in some embodiments, the third solution, which preferably includes EDTA as a chelating agent to remove primarily divalent cations, such as magnesium cations, further includes a MOPS buffer and has a pH value of from 6 to 8. In some embodiments, the third solution may obtained by preparing a MOPS buffer solution, adjusting the pH of said MOPS buffer solution to a range of 1 to 10, preferably 6 to 8, and then adding EDTA as a preferred chelating agent.

In some embodiments, the third solution comprises 40 mM MOPS and 10 mM EDTA, and the pH is from 6 to 8, preferably from 6.5 to 7.5.

The method for purifying mRNA molecules of the present invention further includes step (IIb) of washing the purified mRNA molecules obtained in step (IIa) using a fourth solution. The fourth solution can be any solution in which the mRNA molecules remain stable, that is, they do not undergo hydrolysis. Step (IIb) is advantageous for removing components of the third solution, said fourth solution comprising, for example, sodium chloride or sodium citrate or being water.

As stated above, the second and fourth solutions can be any solution in which the mRNA molecules remain stable, that is, they do not undergo hydrolysis. Such a liquid may be, for example, water, preferably nuclease-free water or RNase-free water such as water for injection (WFI water). In the case of the fourth solution, such liquid may further be, for example, a HEPES-based buffer solution containing glucose, sodium chloride and/or sodium citrate.

Thus, in some embodiments, the fourth solution is water. In some embodiments, the second and fourth solution is water.

In some embodiments, the fourth solution includes sodium chloride and/or citrate.

In some embodiments, the second solution is water and/or the fourth solution is water or includes sodium chloride and/or citrate. The advantage of this formulation is that it provides a solution containing the purified mRNA molecules from step (IIb) without adding components that would have to be removed in additional steps before further downstream processing or use.

In some embodiments, the second solution is water and the fourth solution is water and includes sodium chloride and/or citrate, preferably at a pH in the range of 4 to 5. A significant advantage of such a formulation is primarily therapeutic, since the chloride and/or sodium citrate is preferably contained in a pharmaceutical composition comprising purified mRNA molecules. Thus, this is advantageous from the point of view of subsequent incorporation of the resulting mRNA molecules into pharmaceutical compositions, since sodium chloride and/or citrate are widely used components of pharmaceutical compositions. Thus, the use of a fourth solution containing sodium chloride or citrate, which is a preferred component of pharmaceutical compositions, provides a time-saving and economical integration of the steps necessary to purify said mRNA molecules and incorporate them into a pharmaceutical composition.

In the case of the fourth solution comprising sodium chloride and/or citrate, the pH of the fourth solution is preferably in the range of 3 to 6, more preferably in the range of 3.5 to 5.5, and even more preferably in the range of 4 to 5.

In preferred embodiments of the method of the present invention, ethanol is not included in any of the first, second, third and/or fourth solutions used in the method.

As stated above, it has surprisingly been found that the method of the present invention can achieve a high degree of purification of mRNA molecules. Preferably, the solution obtained by this method includes purified mRNA molecules, but contains no or only very low levels of hydrolysis products and immature mRNA molecules. More preferably, the percentage of mRNA molecules in the resulting solution after step (IIb) is at least 80%, even more preferably at least 90%, even more preferably at least 95% of all mRNA molecules (mRNA molecules, hydrolysis product and immature mRNA molecules). Such content can be measured by methods generally known to those skilled in the art, such as gel electrophoresis, high performance liquid chromatography (HPLC) analysis, or from capillary gel electrophoresis or capillary electrophoresis electropherogram studies, such as Fragment Analyzer analysis. The latter approach is described in more detail in the Examples section below.

In some embodiments of the present invention, at least step (IIa), preferably steps (Ia) to (IIb), is carried out at a temperature in the range of from 0°C to 25°C, preferably from 2°C to 8°C. Thus, the method of the present invention is preferably carried out in the range from 0°C to 25°C at least in the case of step (IIa), that is, the purification of mRNA molecules from the dissolved mRNA molecules obtained in step (Ib) using a third solution comprising a highly effective chelating agent, preferably EDTA. Thus, the immature mRNA molecules and hydrolysis products are surprised when stage (IIa) is carried out at a temperature in the range from 0°C to 25°C, preferably in the range from 2°C to 8°C. The advantage of using particularly low temperatures, such as in the range from 2°C to 8°C, is that the degree of degradation of the mRNA molecules during hydrolysis is reduced compared to the degree of degradation that is observed when especially stage (IIa) is carried out at higher temperatures. temperature, such as above 25°C. Thus, to obtain large quantities of purified mRNA molecules, it is advantageous to carry out at least step (IIa), preferably steps (Ia) to (IIb), of the method of the present invention at a temperature in the range of 0° C to 25°C, preferably in the range from 2°C to 8°C.

In some embodiments, the precipitated mRNA molecules included in the suspension are produced by in vitro transcription, preferably using a DNA template. Thus, the mRNA molecules purified by the method of the present invention can be obtained by any method known in the art. Preferably, the mRNA molecules are transcribed in vitro using a DNA template.

In vitro transcription requires a purified linear or linearized DNA template containing a promoter, ribonucleotide triphosphates, a buffer system and an appropriate RNA polymerase, such as T7 RNA polymerase. For example, such purified linear DNA template can be obtained by chemical synthesis in vitro, optionally by template amplification using, for example, a PCR-based method. In another embodiment, the template for DNA transcription can be obtained from a DNA plasmid, which is generally obtained by cell lysis, purified and linearized, for example, using a site-specific restriction enzyme. In both cases, the resulting linear DNA template sequence can be used to transcribe mRNA molecules in vitro in the presence of A, C, G, T, and U nucleotides using standard laboratory protocols.

In some embodiments, the purified mRNA molecules are produced by in vitro transcription in the presence of unmodified and/or modified nucleotides. In this way, purified mRNA molecules can be synthesized by in vitro transcription from a linear or linearized DNA template using unmodified and/or modified nucleotides. The term "unmodified nucleotide" as used herein refers to nucleotides A, C, G, T and U, as described above. First of all, in the case of in vitro transcription of mRNA molecules, the term refers to nucleotides A, C, G, T and U.

The term "modified nucleotide" as used herein refers to any natural, chemically synthesized isomer of nucleotides A, C, G, T and U, as well as any natural or chemically synthesized analogues, alternative or modified nucleotide or isomer thereof, including, for example, chemical modifications or substituted residues. Modified nucleotides may include modification of a base and/or modification of a carbohydrate moiety. Modified nucleotides may also include modifications to the phosphate group, for example, at the 5' cap of the mRNA molecule. Modified nucleotides may also include nucleotides that are synthesized post-transcriptionally using covalent modification of the nucleotides. In addition, any suitable mixture of unmodified and/or modified nucleotides can be used. An unlimited number of typical modified nucleotides can be found in the literature (for example, see Cantara et al., Nucleic Acids Res, 2011, 39(Issue suppl_1):D195-D201, Helm and Alfonzo, Chem Biol, 2014, 21(2): 174-185, Carell et al., Angew Chem Int Ed Engl, 2012, 51(29): 7110-31) and some preferred modified nucleotides are listed in the following list based on their corresponding nucleoside residue:

1-methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2'-O-ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6 -threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, 2'-O-methyladenosine, N6,N6 ,2'-O-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6-2- methylthio-N6-threonyl carbamoyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 7-methyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, 2'-amino-2'-deoxyadenosine, 2'-azido -2'-deoxyadenosine, 2'-fluoro-2'-deoxyadenosine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine; 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-hydroxycytidine, lysidine, N4-acetyl-2'-O-methylcytidine, 5-formyl-2 '-O-methylcytidine, 5,2'-O-dimethylcytidine, 2-O-methylcytidine, N4,2'-O-dimethylcytidine, N4,N4,2'-O-trimethylcytidine, isocytidine, pseudocytidine, pseudoisocytidine, 2-thio -cytidine, 2'-methyl-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-bromocytidine, 2'-azido-2'-deoxycytidine , 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-aza-cytidine, 3-methyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio -5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, 2-methoxy -cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio -zebularine, 2-thio-zebularine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, guanosine 2'-O-ribosylphosphate, 7-methylguanosine, hydroxyvibutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7 -deazaguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, N2,2'-O-dimethylguanosine, 1,2'-O-dimethylguanosine, 2'-O-methylguanosine, N2,N2,2 '-O-trimethylguanosine, n2, n2j-trimethylguanosine, isoguanosine, 4-demethylviosin, epoxycovosine, not fully modified hydroxyvibutosine, methylated not fully modified hydroxyvibutosine, isuviosin, peroxivibutosine, galactosyl-kevosin, mannozyl-keys, sivosin, bibu, vibu Tozin, methylviosin , viosine, 7-aminocarboxypropyldemethylviosine, 7-aminocarboxypropylviosine, 7-aminocarboxypropylviosine methyl ester, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6 -thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, 8-oxo-guanosine, 7 -methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, N1-methylguanosine, 2'-amino- 3'-deoxyguanosine, 2'-azido-2'-deoxyguanosine, 2'-fluoro-2'-deoxyguanosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine , 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3, 2'-O-dimethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2-thiouridine, 5-methoxycarbonylmethyl-2'-O- methyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyluridine, 2'-O-methyl-2-thiouridine, 2-thio-2'-O -methyluridine, uridine 5-hydroxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-hydroxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5 -methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2'-O-methylpseudouridine, 5 -formyluridine, 5-aminomethyl-2-geranyluridine, 5-taurinomethyluridine, 5-ioduridine, 5-bromuridine, 2'-methyl-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2' -deoxyuridine, 2'-fluoro-2'-deoxyuridine, inosine, 1-methylinosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 5-aza-uridine, 2-thio-5-aza-uridine , 4-thio-pseudouridine, 2-thio-pseudouridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio -uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudo-uridine, 4-thio-1-methyl-pseudo-uridine, 2-thio-1-methyl-pseudo-uridine, 1-methyl-1 -deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy -pseudouridine, 4-methoxy-2-thio-pseudouridine, 1,2'-O-dimethyladenosine, 1,2'-O-dimethylguanosine, 1,2'-O-dimethylinosine, 2,8-dimethyladenosine, 2-methylthiomethylenethio- N6-isopentenyl-adenosine, 2-geranylthiouridine, 2-lysidine, 2-methylthio cyclic N6-threonylcarbamoyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine , 2-selenouridine, 2-thio-2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylinosine, 2'-O-methylpseudouridine , 2'-O-methyluridine, 2'-O-methyluridine 5-hydroxyacetic acid methyl ester, 2'-O-ribosyladenosine phosphate, 2'-O-ribosylguanosine phosphate, 3,2'-O-dimethyluridine, 3-(3-amino -3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 5,2'-O-dimethylcytidine, 5,2'-O-dimethyluridine, 5-(carboxyhydroxymethyl) methyl ester -2'-O-methyluridine, 55-(isopentenylaminomethyl)-2'-O-methyluridine, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylmethyl-2'-O- methyluridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-cyanomethyluridine, 5-formyl-2 '-O-methylcytidine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxylpropyl-demethylviosine, 7-methylguanosine, 8-methyladenosine, N2,2'-O-dimethylguanosine, N2, 7,2'-O-trimethylguanosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2,7-trimethylguanosine, N4,2'-O-dimethylcytidine , N4,N4,2'-O-trimethylcytidine, N4,N4-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6 -formyladenosine, N6-hydroxymethyladenosine, agmatidine, 2-methylthio cyclic N6-threonylcarbamoyladenosine, glutamyl-quevosine, guanosine added to any nucleotide, guanylated 5' end, hydroxy-N6-threonylcarbamoyladenosine, most preferably pseudouridine, N1-methyl-pseudouridine, 2 '-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-methylcytidine, 2-thiouridine, 5-ioduridine and/or 5-methyluridine.

Moreover, the term "modified nucleotide" includes nucleotides containing isotopes such as deuterium. The term "isotope" refers to an element containing an equal number of protons but a different number of neutrons, resulting in different mass numbers. Thus, isotopes of hydrogen, for example, are not limited to deuterium, but also include tritium. Moreover, the mRNA molecule may also contain isotopes of other elements, including, for example, isotopes of carbon, oxygen, nitrogen and phosphorus. It is also possible that the modified nucleotides are deuterated or contain another isotope of hydrogen or oxygen, carbon, nitrogen or phosphorus.

Therefore, in the case where the purified mRNA molecules are produced by in vitro transcription in the presence of four types of nucleotides, i.e. nucleotides A, C, G and U, the total number of types of modified nucleotides may be 0, 1, 2, 3 or 4. Thus , in some embodiments, the modified nucleotide may be at least one nucleotide of one type, such as at least one U nucleotide. In some embodiments, the modified nucleotide may be at least one nucleotide of a total of two types, such as at least one U nucleotide and at least one C nucleotide. In some embodiments, the modified nucleotide may be at least one nucleotide of all three types, such as at least one G nucleotide, at least one U nucleotide, and at least one C nucleotide. In some embodiments, the modified the nucleotide may be at least one nucleotide of all four types. In all of these embodiments, one or more nucleotides of the same type may be modified, with the percentage of said modified nucleotides being 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.

In some embodiments, the total percentage of modified nucleotides included in the purified mRNA molecules is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.

The purified mRNA molecules can, for example, be characterized as follows: from 0.5 to 50%, preferably from 5 to 50% of the U nucleotides, and from 5 to 50% of the C nucleotides are modified. Said modified U nucleotides are preferably 5-iodouridine, and said modified C nucleotides are preferably 5-iodocytidine.

In some embodiments, the purified mRNA molecules may, for example, be characterized as follows: from 15 to 25% of the U nucleotides and from 5 to 15% of the C nucleotides are modified, wherein the modified U nucleotides are preferably 5-methyluridine and the modified C nucleotides are preferably 5-methyluridine. preferably 5-iodocytidine.

In some embodiments, the purified mRNA molecules may, for example, be characterized as follows: from 30 to 50% of the U nucleotides and from 10 to 20% of the C nucleotides are modified, wherein the modified U nucleotides are preferably 5-ioduridine and the modified C nucleotides are preferably 5-ioduridine. preferably 5-iodocytidine.

In some embodiments, the purified mRNA molecules may, for example, be characterized as follows: from 30 to 50% of the U nucleotides and from 5 to 15% of the C nucleotides are modified, wherein the modified U nucleotides are preferably 5-ioduridine and the modified C nucleotides are preferably 5-ioduridine. preferably 5-iodocytidine.

In some embodiments, the purified mRNA molecules may, for example, be characterized as follows: from 0.5 to 5% of the U nucleotides and from 25 to 35% of the C nucleotides are modified, wherein said modified U nucleotides preferably are 2-thiouridine, and said modified The C nucleotides are preferably 5-methylcytidine.

The purified mRNA molecules can, for example, be characterized as follows: from 50 to 100%, preferably 100%, of the U nucleotides are modified, wherein said modified U nucleotides are preferably Nl-methylpseudouridine.

In some embodiments of the present invention, the method prior to step (Ia) includes the step of preparing a suspension comprising precipitated mRNA molecules using ammonium acetate. Precipitation of mRNA molecules has the advantage of removing salts, nucleotides and proteins, primarily because mRNA precipitation is a simple and cost-effective method. Various precipitation solutions are known to those skilled in the art, including, for example, GSCN, NH ₄ OAc, ethanol, lithium chloride, sodium acetate and sodium chloride. However, specifically in relation to therapeutic applications, a significant advantage lies in the removal of salts such as GSCN and LDS. Thus, in some embodiments, the mRNA molecules are precipitated using NH ₄ OAc. Thus, the suspension for precipitation of mRNA molecules used in step (Ia) is preferably prepared using NH ₄ OAc.

As noted above, in some embodiments, the first solution contains ammonium acetate. Preferably, the total amount of NH ₄ OAc in the suspension and first solution is in the range from 0.1 mol/L to 5 mol/L, preferably in the range from 1 mol/L to 4 mol/L, more preferably from 2 mol/L to 3 mol/l, and thus less than 5 mol/l, preferably less than 4 mol/l, more preferably less than 3 mol/l. The low amount of ammonium acetate is a particular advantage in terms of the duration and cost-effectiveness of further processing. The first solution may furthermore have a pH value in the range from 1 to 12, preferably from 6.5 to 7.5.

In some embodiments, the first solution contains ammonium acetate, wherein the total amount of NH ₄ OAc in the suspension and in the first solution is in the range of 1 mol/L to 4 mol/L, preferably in the range of 2 mol/L to 3 mol/L.

In some embodiments, the pH of the first solution is in the range of from 1 to 12, preferably from 6.5 to 7.5, and said solution contains ammonium acetate, wherein the total amount of NH ₄ OAc in the suspension and in the first solution is in the range of 1 mole /l to 4 mol/l, preferably in the range from 2 mol/l to 3 mol/l.

In some embodiments, the method for purifying the mRNA molecules further includes adding a fifth solution to the dissolved mRNA molecules obtained in step (Ib) before purifying the mRNA molecules using a third solution including the chelating agent of step (IIa). The fifth solution preferably includes the same chelating agent as in the third solution. Thus, the fifth solution preferably includes EDTA as a chelating agent to remove primarily divalent cations, such as magnesium cations, and optionally a MOPS buffer. However, the concentration of the chelating agent included in the fifth solution is preferably higher than the concentration of the chelating agent included in the third solution. By adding a fifth solution to the dissolved mRNA molecules obtained in step (Ib) before performing step (IIa), the composition of the solution in which the mRNA molecules obtained in step (Ib) are suspended can be adjusted to the composition of the third solution used in step ( IIa). The advantage of such an operation is that possible dilution effects can be reduced, and thus the throughput and/or reproducibility of purification of an mRNA molecule can be increased according to the method described in this context.

In some embodiments, the method for purifying mRNA molecules further comprises dephosphorylation and/or polyadenylation and/or post-capping of the mRNA molecules. Thus, mRNA molecules can not only be purified by the method of the present invention, but also further modified to ensure their ability to be translated into functional amino acid sequences, such as proteins. Such modifications include dephosphorylation of mRNA molecules to remove, for example, 5'-mono-, di- and/or triphosphates that were added during in vitro synthesis and/or cotranscriptional 5' capping, polyadenylation of mRNA molecules, since the tail poly(A) in most cases is the main factor determining the lifetime of an mRNA molecule, and post-capping of mRNA molecules if co-transcriptional 5'-capping was not performed. Since primarily 5'-caps and 3'-poly(A) tails are considered to be the main factors determining the effectiveness of mRNA, the inclusion of such features in the method for purifying an mRNA molecule is advantageous in terms of maximizing the time and cost-effectiveness of processing the mRNA molecule and maximizing the duration of action purified mRNA molecules.

The advantage of having a 5' cap is to increase the stability of the mRNA molecules and thus increase the duration of action of the mRNA molecules. Thus, if the precipitated mRNA molecules to be purified do not contain a 5' cap or a 5' cap analogue, the mRNA molecules are preferably subjected to post-transcriptional 5' capping, for example by enzymatic incorporation of a C1-m7G cap or an m7GpppG cap. However, more preferably, the mRNA molecules are subjected to post-transcriptional 5' capping, for example using an ARCA cap analog or using Trilink technology (CleanCap technology, see US 2018/273576 A1). In this case, the advantage of using the TFF method of the present invention is the removal of pure cap analogue.

Moreover, in the event that the precipitated mRNA molecules to be purified do not contain a 3'-poly(A) tail, the mRNA molecules are preferably enzymatically polyadenylated using a poly(A) tail that contains at least 100 nucleotides of A, preferably at least 120 nucleotides A, more preferably at least 240 nucleotides of A. Optionally, the added poly(A) tail includes non-A nucleotides, preferably G nucleotides in the poly(A) tail sequence and/or in one terminal fragment of the poly(A) tail, wherein the specified terminal fragment is not included in the 3'-terminal fragment of the purified mRNA molecules. It has been found that the advantage of adding a poly(A) tail to mRNA molecules is to increase the duration of action of said mRNA molecule and thus achieve the required levels of translation.

Therefore, in some embodiments, the method for purifying tRNA molecules of the present invention further comprises dephosphorylation of the mRNA molecules.

In other embodiments, the method further includes polyadenylation of mRNA molecules.

In yet other embodiments, the method further includes post-capping the mRNA molecules.

In some embodiments, a method for purifying mRNA molecules includes at least dephosphorylation and polyadenylation of the mRNA molecules.

In some embodiments, the method for purifying mRNA molecules of the present invention includes at least dephosphorylation and post-capping of the mRNA molecules, wherein dephosphorylation of the mRNA molecules occurs after post-capping of the mRNA molecules.

In some embodiments, a method for purifying mRNA molecules includes dephosphorylation of the mRNA molecules obtained in step (Ib), followed by steps (Ia)-(IIb), followed by polyadenylation of the resulting mRNA molecules, and then repeated steps (Ia)-(IIb) wherein the last step (Ia) is carried out at a temperature in the range from 20°C to 30°C, preferably in the range from 23°C to 27°C, more preferably at 25°C, optionally followed by filtration of the resulting concentrate comprising the mRNA molecules . Thus, to ensure maximum cost-effectiveness and reduce the time required for processing mRNA molecules, the method for purifying mRNA molecules additionally includes the stages of dephosphorylation and polyadenylation of mRNA molecules. First of all, the present inventors have found that it is advantageous to combine the last two steps with additional purification steps of the mRNA molecules to also remove components introduced during dephosphorylation and polyadenylation.

Thus, primarily in the case where the mRNA molecules to be purified include a cotranscriptionally included 5' cap or 5' cap analogue, the method for purifying the mRNA molecules includes the following steps. First, the precipitated mRNA molecules are purified from the suspension comprising said precipitated mRNA molecules using the first solution in step (Ia), said first solution preferably comprising ammonium acetate. The purified precipitated mRNA molecules in step (Ib) are then washed and dissolved using a second solution, preferably water, to remove, for example, proteins, salts and nucleoside triphosphates. The dissolved mRNA molecules are then dephosphorylated by methods known to those skilled in the art to remove 5' mono-, di- and triphosphates from mRNA molecules that may not have been capped. The dephosphorylated mRNA molecules are purified using a first solution, preferably comprising ammonium acetate, in a further step (Ia) and then washed using a second solution, which is preferably water, in a further step (Ib). The mRNA molecules thus obtained are then purified in step (IIa) using a third solution comprising a chelating agent, preferably EDTA, and preferably MOPS at a pH ranging from 0 to 8, more preferably at a pH ranging from 6.5 to 7.5 , to remove immature mRNA molecules and hydrolysis products. The third solution is then removed during the washing step of step (IIb) using the fourth solution. If a polyadenylation step is not required, for example because a DNA template that includes a sequence encoding a poly(A) tail used for in vitro transcription, the resulting purified mRNA molecules are washed in step (IIb) using a fourth solution, preferably comprising sodium citrate and/or chloride, and said step (IIb) is optionally followed by an additional filtration step using, for example, a filtration membrane with a pore size of less than 0.3 μm. In case the polyadenylation step is preferred, for example because a DNA template was used for in vitro transcription that does not contain a sequence encoding the poly(A) tail, the resulting purified mRNA molecules are washed in step (IIb) using a fourth solution, preferably water, and can then be extended by enzymatically incorporating the 3'-poly(A) tail using standard laboratory protocols to increase the longevity of the mRNA molecules, such as the addition of poly(A) polymerase. The mRNA molecules are then treated in steps (Ia) to (IIb) as described above, except that step (Ia) is carried out at a temperature in the range of 20°C to 30°C, preferably in the range of 23°C to 37°C, more preferably at 25°C, for example to remove added poly(A) polymerase, and except that the fourth solution used in step (IIb) preferably includes sodium citrate or chloride, to obtain purified mRNA molecules , including 5' caps or analogues of 5' caps or 3' poly(A) tails. Optionally, the resulting purified mRNA molecules can be subjected to an additional filtration step, for example using a filtration membrane with a pore size of less than 0.3 μm.

In the event that the mRNA molecules to be purified do not include a co-transcriptionally incorporated 5' cap or 5' cap analog, the method for purifying the mRNA molecules of the present invention, compared to the procedure described above, includes an additional step, preferably the post-transcriptional addition of a 5' cap or analog 5' cap in the presence of an enzyme. Thus, a method for purifying mRNA molecules may include the following steps. First, the precipitated mRNA molecules are purified from a suspension that includes said precipitated mRNA molecules using the first solution in step (Ia), said first solution preferably comprising ammonium acetate. The purified precipitated mRNA molecules are then washed in step (Ib) and dissolved using a second solution, preferably water, to remove, for example, proteins, salts and nucleoside triphosphates. The method additionally includes capping the mRNA molecules obtained at stage (Ib), and then carrying out stages (Ia) and (Ib), dephosphorylation of the resulting mRNA molecules, followed by stages (Ia)-(IIb). If the polyadenylation step is not required, for example because a DNA template that includes the sequence that encodes the poly(A) tail has been used for in vitro transcription, the resulting purified mRNA molecules are washed in step (IIb) using the fourth solution preferably comprising sodium citrate and/or chloride, and said step (IIb) is optionally followed by a further filtration step using, for example, a filtration membrane with a pore size of less than 0.3 μm. In the case where a polyadenylation step is appropriate, for example because a DNA template was used for in vitro transcription that does not include a sequence that encodes the poly(A) tail, the resulting purified mRNA molecules are washed in step (IIb) with using a fourth solution, preferably water, and can then be extended by adding a 3' poly(A) tail in the presence of an enzyme using a standard laboratory protocol to extend the duration of the mRNA molecules, such as the addition of poly(A) polymerase. The mRNA molecules are then processed in steps (Ia) to (IIb), where step (Ia) is preferably carried out at a temperature in the range of 20°C to 30°C, preferably in the range of 23°C to 37°C, more preferably at 25°C, for example, to remove the added poly(A) polymerase, and then step (IIb) is carried out using a fourth solution, preferably comprising citrate and/or sodium chloride, optionally followed by filtration of the resulting concentrate containing the mRNA molecules.

Thus, steps (Ia) to (IIb) are preferably carried out before any extension of the mRNA molecules, such as 3' polyadenylation and optionally 5' capping, occurs. First of all, steps (Ia)-(IIb) are preferably carried out before 3' polyadenylation of the mRNA molecules, and steps (Ia)-(Ib) are preferably carried out before 5' capping. This approach is used in all four embodiments of the present invention, that is, for the purification of mRNA molecules that have been subjected to co-transcriptional and post-transcriptional 5' capping, respectively.

Moreover, in all four embodiments described above, after the steps of dephosphorylation of the mRNA molecules, steps (Ia) and (Ib) are optional. Therefore, some embodiments correspond to the four embodiments as described above, except that the steps of dephosphorylation of the mRNA molecules are followed by steps (Ia) and (Ib).

TFF filtration can be carried out using a capsule, cassette and cassette holder or hollow fiber module as the filtration device. First of all, hollow fiber modules are fully pre-assembled, pre-sterilized, disposable flow channels that enable sterile processing and provide a cost-effective method of packaging filtration membranes.

Filtration membranes used to purify mRNA molecules can be any type of material that is suitable for purifying mRNA molecules and therefore does not interact with the mRNA molecules being purified. Typical filtration membrane materials include unmodified polyethersulfone (PES), modified polysulfone (mPES), mPES hollow fiber membrane, polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, mixed cellulose ether (ME), ultra-high molecular weight polyethylene (UPE) , polyfluorotetraethylene (PTFE), nylon, polysulfone (PS), polyacrylonitrile, polypropylene, polyvinyl chloride, polyvinylidene difluoride (PVDF) and combinations thereof.

Filtration membranes are characterized by a molecular weight cutoff value (MWCO), which is the lowest molecular weight of particles in daltons of which 90% are retained above the membrane. Preferably, the pore size of the filtration membrane ensures that the mRNA molecules are retained while smaller components, i.e., less than MWCO, pass through the filtration membrane and are called filtrate. Since different components of different sizes are removed, it is advantageous to select the pore size of the filtration membrane in accordance with the size of the components that need to be removed at the appropriate stage of the method of the present invention.

In some embodiments of the present invention, in the case of step (Ia), a filtration membrane with a MWCO cutoff in the range of 300 kDa to 0.65 μm, preferably 500 kDa, is used to filter the TFF, and/or in the case of steps (Ib) and (IIb ) using a filtration membrane with a MWCO cutoff in the range of 1 kDa to 0.65 μm, preferably 1 kDa to 300 kDa, more preferably 1 kDa to 50 kDa, even more preferably 50 kDa, 70 kDa and/or 100 kDa, and/or in the case of step (IIa), use a filtration membrane with a cutoff limit of at least 50 kDa, preferably at least 70 kDa, more preferably 70 kDa or 100 kDa. The filtration membrane is important because the pore size of the filtration membrane determines the size of particles, such as mRNA molecules and its components, that can pass through the filtration membrane and thus be contained in the filtrate.

Thus, in the case of step (Ia), the MWCO of the filtration membrane is preferably 300 kDa or 0.065 μm. Preferably, the MWCO value is selected from the group consisting of 300 kDa, 500 kDa, 750 kDa, 0.05 kDa, 0.1 μm, 0.2 μm, 0.45 μm, 0.5 μm, 0.65 μm, and The MWCO is particularly preferably 500 kDa. The advantage is to retain precipitated mRNA molecules while removing, for example, enzymes and proteins contained in cells that have been lysed to obtain purified mRNA molecules or in the case of in vitro transcribed enzyme mRNA molecules, modified and unmodified nucleoside triphosphates, 5' caps, 5' cap analogs and buffer components contained in in vitro transcription reaction mixtures. Thus, the appropriate MWCO value can be selected taking into account, for example, the size of enzymes used in previous processing steps, such as in vitro transcription of purified mRNA molecules, and which need to be removed by TFF filtration in step (Ia). T7RNA polymerase is a well-known enzyme in the art that is not effectively removed when using a filtration membrane with an MWCO value of less than 100 kDa. When using a membrane with a MWCO value ranging from 1 kDa to 750 kDa in step (Ia), TFF filtration is called ultrafiltration, while filtration using a membrane with a MWCO value ranging from 0.05 µm to 0.65 µm is TFF filtration called microfiltration.

In the case of steps (Ib) and (IIb), the MWCO value is preferably at least 1 kDa, preferably 1 kDa to 50 kDa, more preferably at least 50 kDa, and even more preferably at least 70 kDa. Preferably, the MWCO value is selected from the group consisting of 1 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa, 100 kDa, 300 kDa, 0.05 μm, 0.1 μm, 0.2 μm, 0.45 μm, 0.5 μm and 0.65 μm, and the MWCO value is especially preferably 50 kDa, 70 kDa or 100 kDa. Such features are advantageous for the purification of mRNA molecules, due to the effective removal of salts and other components contained in the corresponding solution, such as ammonium acetate, chelating agents such as EDTA, a buffer such as MOPS, immature mRNA molecules and/or products hydrolysis. In the case of relatively large mRNA molecules, an MWCO value of more than 300 kDa can also be considered.

In the case of step (IIa), the MWCO value is preferably at least 50 kDa, more preferably at least 70 kDa, even more preferably 70 kDa or 100 kDa. When using, for example, a MOPS-EDTA buffer solution as described above, a MWCO value of 100 kDa is particularly preferred. In the case of other highly effective chelating agents or other highly effective combinations of chelating agent and buffer, even MWCO values of 100 kDa or greater may be considered for the purification of relatively large mRNA molecules.

Moreover, processes using TFF filtration can be characterized in terms of various variables, the most important of which are transmembrane pressure and flow rate.

The term "transmembrane pressure" (TMP) refers to the driving force that ensures the movement of components through the filtration membrane. In some embodiments, the transmembrane pressure in the case of steps (Ia) and (Ib), for example, on a laboratory scale, typically 500 mg of mRNA, is from 100 mbar to 500 mbar, preferably from 200 mbar to 400 mbar, and in the case of steps (IIa ) and (IIb) from 2 mbar to 20 mbar, preferably from 5 mbar to 15 mbar.

The term "flow" refers to the volume of solution that flows through the TFF system and primarily through the area of the filtration membrane, and "flow rate" refers to the volume of solution flowing through said system during a given time. Thus, flow rate or lateral velocity refers to the rate at which a solution flows through the filtration membrane. In some embodiments, the flow rate for steps (Ia) and (Ib) is, for example, 500 mg of mRNA, typically on a laboratory scale, from 1.5 L/min to 2.5 L/min, preferably from 1.6 l/min to 1.9 l/min, and in the case of stages (IIa) and (IIb) from 0.2 l/min to 0.6 l/min, preferably from 0.35 l/min to 0.45 l /min.

In the most preferred embodiments of the present invention, the method for purifying mRNA molecules is carried out using continuous TFF filtration. The term continuous TFF refers to a TFF system in which the concentrate is used as a feed stream in another TFF cycle. As used herein, the term “feed stream” means a solution or suspension comprising the mRNA molecules to be purified. Therefore, the original feed stream comprising the mRNA molecules is subjected to a first round of filtration using TFF, and the resulting concentrate comprising the mRNA molecules is again used as a feed stream for another cycle of TFF while circulating at least the mRNA molecules. This combination is advantageous for increasing purification levels in repeated automated purification steps, as well as reducing the risk of loss of mRNA molecules as they move through systems.

Thus, in some embodiments, the mRNA molecules are contained in the concentrate after tangential flow filtration, preferably at least in the concentrate obtained in steps (Ia)-(IIa). In the case of stage (IIb), the mRNA molecules can be contained in the concentrate or in the filtrate.

In some embodiments, the mRNA molecules are contained in the concentrate obtained in steps (Ia) through (IIb).

In some embodiments, the concentrate obtained in step (Ia) is used as the solution feed stream for tangential flow filtration in step (Ib), the concentrate obtained in step (Ib) is used as the solution feed stream in stage (IIa), and the concentrate obtained in stage (IIa) as the feed solution in stage (IIb). Thus, the present invention is preferably carried out using continuous TFF filtration to perform at least steps (Ia) to (IIb). In more detail, for the purification of the present invention, the mRNA molecules are preferably circulated in a continuous TFF system. Thus, in one embodiment, at least steps (Ia)-(IIa), preferably steps (Ia)-(IIb), are carried out using continuous TFF.

An additional advantage of the TFF system is that it can be used in a simple manner for diafiltration and especially for continuous diafiltration. Using diafiltration, parts of a solution or suspension can be replaced with others. For example, in intermittent diafiltration, the solution is first diluted and then concentrated again to its original volume. However, this process may have a negative effect on the functionality of the purified mRNA molecules.

Therefore, in preferred embodiments, the mRNA molecules to be purified are circulated through at least steps (Ia) to (IIb), in a continuous TFF system for purification in the manner described above using a constant volume of diafiltration. Constant volume or continuous diafiltration refers to a filtration method where a constant volume is maintained in the filtration system. Thus, the same amount of solution or suspension is added as a volume of solution or suspension that passes through the filtration membrane as filtrate. Thus, the volume of concentrate is equal to the volume that was initially supplied to the TFF system, that is, the feed volume and the volume for washing, that is, the diafiltration volume, is equal to the volume of filtrate. The combination of continuous TFF and continuous diafiltration is advantageous in terms of removing contaminants as a filtrate while keeping the purified mRNA molecules in the circulating portion of the TFF system at a constant volume to maintain their function.

Therefore, in some embodiments, at least steps (Ia)-(IIa), preferably steps (Ia)-(IIb), are carried out using diafiltration, preferably continuous diafiltration.

In some embodiments, the diafiltration volume of any first, second, third, and/or fourth solution is at least 1 times the volume of the suspension of step (Ia), e.g., 1 times, 2 times, 3 times, 4 times, 5 times multiple, preferably at least 10 times the volume. Determining the specified diafiltration volume is of particular interest from the point of view of the optimal compromise between time costs, cost-effectiveness and the level of purification that needs to be achieved. It has been found that the diafiltration volume of any of the first, second, third and/or fourth solution is at least 1 times the volume of the suspension of step (Ia), preferably at least 10 times. The advantage is to achieve significant component removal while still providing high throughput processing, and thus purification of mRNA molecules on a preparative scale.

In some embodiments, the volume of diafiltration is 1 times the volume of the suspension of step (Ia), and thus the volume of the suspension comprising the precipitated mRNA molecules originally introduced into the TFF system in step (Ia).

In some embodiments, the volume of diafiltration is twice the volume of the suspension. In some embodiments, the volume of diafiltration is 3 times the volume of the suspension. In some embodiments, the volume of diafiltration is 4 times the volume of the suspension. In some embodiments, the volume of diafiltration is 5 times the volume of the suspension. In some embodiments, the volume of diafiltration is at least 5 times the volume of the suspension in step (Ia).

In some embodiments, the volume of diafiltration is at least 10 times the volume of the suspension in step (Ia), and therefore the volume of the suspension, including precipitated mRNA molecules, originally introduced into the TFF system in step (Ia). It has been found that using a diafiltration volume of at least 10 times the volume initially introduced into the TFF system is advantageous for significant removal of components used in the corresponding step as described above.

Therefore, it is preferable that the diafiltration volume of any first, second, third and/or fourth solution is at least 10 times the volume of the suspension of step (Ia). Even more preferably, at least the diafiltration volume of the third and fourth solution is at least 10 times the volume of the suspension in step (Ia).

In some embodiments, the volume of diafiltration of the first, second, third and fourth solution is 10 times the volume of the suspension in step (Ia).

A typical fully automated, closed-loop system for obtaining purified mRNA molecules according to the method of the present invention is shown in FIG. 2. The system includes a continuous TFF system shown in FIG. 1, which is in fluid communication with the mRNA production system, preferably through a first valve. It should be noted that the term "valve" as used herein includes microprocessor-controlled (eg, two- and/or three-way) shut-off valves, clamps and connectors, preferably MPC valves and/or connectors. Moreover, it is preferable that the MPC connector be used in combination with a clamp. Thus, two elements, for example two hoses, can be connected in such a way as to ensure single use and/or compatibility with various systems, resistance to high pressure and/or changes in pressure, for example in one of the hoses, and/or low cost.

The mRNA production system may include a reaction vessel and at least one other vessel. The reaction vessel and at least one additional vessel are preferably in fluid communication with each other and with the TFF system via a connecting hose. The first valve is located between the connecting hose of the mRNA production system and the hose of the TFF system, preferably the hose in which the feed flow circulates from the reservoir to the filtration device. In another embodiment, the mRNA production system may be in fluid connection with a hose in which the concentrate is circulated from the filtration device to the reservoir. It should be noted that as used herein, the term “hose” can refer to a hose, pipe, conduit, or a combination thereof. Moreover, the connecting hose of the mRNA production system may include an auxiliary pump. The advantage of the auxiliary pump is that it automatically adjusts the flow rates passing through the connecting hose.

The purified mRNA molecules according to the method of the present invention can be obtained, for example, by culturing cells to amplify the mRNA molecules in the cell, or preferably under in vitro transcription (IVT) conditions. In the case of producing mRNA molecules from cells, the cells, such as E. coli cells, can be cultured in a fermenter under cell-specific culture conditions suitable for producing the mRNA molecules. The parameters of said culture conditions are preferably automatically controlled, for example by providing appropriate quantities of culture medium, gas and/or buffer substance. Therefore, a system for producing mRNA molecules may include a reaction vessel and at least one additional vessel, where the reaction vessel is a fermenter and the at least one additional vessel includes, for example, an appropriate culture medium, gas and/or buffer solution.

Alternatively, if the mRNA molecules to be purified are produced by the IVT method, an IVT template is required, such as an appropriate DNA template for IVT transcription of the target mRNA molecules being purified, and IVT reagents, such as T7 RNA polymerase, and a nucleotide triphosphate (NTP) mixture. Therefore, an mRNA production system may include a reaction vessel and at least one additional vessel, wherein the at least one additional vessel includes at least one of said IVT reagents.

Thus, the reaction vessel is preferably in fluid communication with at least one additional vessel, preferably with at least the first additional vessel and the second additional vessel.

The first additional vessel may include at least one of these IVT reagents and is preferably in fluid communication with the reaction vessel through a connecting hose and a second and third valve. The second valve is preferably located between the reaction vessel and the connection hose, and the third valve is located between the connection hose and the first additional vessel. When the first valve closes and the second and third valves open, production of mRNA molecules can occur, for example by the IVT method, preferably in a reaction vessel. When the third valve closes and the first and second valves open, the produced mRNA molecules can be transferred from the mRNA production system to the TFF system.

The second additional vessel may include a solution for precipitating the mRNA molecules, wherein said solution preferably comprises ammonium acetate (NH ₄ OAc), for example 5 M NH ₄ OAc. Preferably, the second additional container is in fluid connection with the connecting hose through the fourth valve. When the second and fourth valves are opened and the first and third valves are closed, the target molecules can be precipitated, preferably in the reaction vessel, or if the reaction vessel is a fermenter, in a second additional vessel. Then, when the first valve is opened, the precipitated mRNA molecules in suspension can be transferred from the mRNA production system to the TFF filtration system for purification of the mRNA molecules by the method of the invention.

Therefore, the system shown in FIG. 2 includes a TFF system, preferably a continuous reservoir diafiltration system. Preferably, the reservoir is in fluid communication with at least three additional vessels, that is, at least a third additional vessel, a fourth additional vessel and a fifth additional vessel, through at least a fifth, sixth and seventh valves, respectively. The third additional container may include the first solution, the fourth additional container the second solution, and the fifth additional container the third solution of the present invention. If the second and fourth solutions are different from each other, the reservoir is preferably in fluid connection with a sixth additional reservoir including the fourth solution, for example through an eighth valve. Therefore, it is preferable that the third additional container includes a first solution, the first solution comprising NH ₄ OAc, for example 2.5 M NH ₄ OAc, to purify the precipitated mRNA molecules, the fourth additional container contains water, for example, water for injection (WFI ) as a second solution, and the fifth additional container is a third solution, wherein the third solution includes EDTA as a chelating agent and optionally MOPS. In case the fourth solution is not water, the sixth additional container preferably includes sodium chloride and/or citrate. Moreover, an auxiliary pump may be present to automatically adjust the flow rates transferred from any of the at least three additional containers to the reservoir.

Therefore, the reservoir preferably includes precipitated mRNA molecules in suspension, which is transferred from the mRNA production system through the first valve when the first valve is opened for a predetermined time. By opening the fifth valve for a predetermined time, the first solution can be added to the suspension of precipitated mRNA molecules, and the mixture can then be transferred to at least one cycle of continuous TFF filtration in a filtration device, the filtration membrane preferably having a molecular weight cutoff of 300 kDa up to 0.65 µm. Thus, the suspension comprising the precipitated mRNA molecules can be purified, for example, from components contained in the IVT mixture, such as proteins, salts and/or NTPs according to step (Ia) of the method described in this context. The suspension comprising the purified precipitated mRNA molecules is preferably transferred back to the reservoir as a concentrate. To carry out step (Ib), that is, washing and dissolving the purified precipitated mRNA molecules obtained in step (Ia), the sixth valve is opened for a predetermined time. Thus, the second solution can be added to the suspension containing the precipitated mRNA molecules present in the reservoir. By performing at least one cycle of continuous filtration of the TFF using a filtration device, preferably a filtration membrane with a cutoff limit of 1 kDa to 0.65 μm, for example, the NH ₄ OAc included in the first solution can be removed. By opening the seventh valve for a predetermined time, the third solution can then be added to the washed and dissolved mRNA molecules in the reservoir. By performing at least one cycle of continuous filtration of TFF using a filtration device, if the filtration membrane has a cutoff limit of 50 kDa, more preferably 100 kDa, immature mRNA molecules and hydrolysis products of mRNA molecules can be effectively removed in step (IIa) of the method described herein context. Step (IIb) can then be carried out to wash the purified mRNA molecules obtained in step (IIa), for example to remove components of the third solution. Therefore, either the sixth or eighth valve is opened for a predetermined time to add either water or a fourth solution comprising sodium chloride and/or citrate to the purified mRNA molecules over at least one cycle of continuous TFF using a filtration device including a filtration filter. a membrane preferably having a cutoff limit of 50 kDa.

Optionally, an additional step is carried out between steps (Ib) and (IIa). Therefore, the TFF system may further include a seventh additional vessel that is in fluid communication with, for example, a reservoir, preferably through a ninth valve. Said seventh additional container may include a fifth solution, wherein the fifth solution preferably includes at least a chelating agent in the third solution, preferably EDTA, at a higher concentration than in said third solution. By opening the ninth valve for a predetermined time after step (Ib), the fifth solution can be added to the mRNA molecules dissolved in the second solution. The advantage of this operation is that it can increase the efficiency and reproducibility of the method of the present invention by reducing the time required to adjust the concentration of at least the chelating agent in the TFF system to the appropriate concentration, as specified herein for the third solution. Moreover, an auxiliary pump may be present to automatically control the flow rates transferred from the seventh additional vessel to the reservoir (not shown).

Optionally, the mRNA molecule production system may further include additional capacity, preferably in fluid connection to the connecting hose of the mRNA molecule production system, preferably through an intermediate valve. The intermediate container may preferably be a sterile bag. By opening the first valve and the intermediate valve for a predetermined time, at least a portion of the suspension or solution comprising the mRNA molecules can be transferred from the TFF system to the mRNA molecule production system and/or from the mRNA molecule production system to the TFF system. The advantage of such an operation is that at least a portion of said suspension or solution is retained, preferably for a predetermined time. Thus, at least said portion of said suspension or solution can be subjected to an additional purification cycle, for example in steps (Ia) and (Ib), in steps (IIa) and (IIb), and/or a combination thereof. In addition, while at least a portion of the suspension or solution is stored in the intermediate container, the TFF system and/or the mRNA molecule production system can be cleaned, washed with water and/or buffer solution, and/or sterilized. Optionally or alternatively, the intermediate vessel can be used to obtain at least a portion of the solution or suspension contained in the intermediate vessel, preferably a solution or suspension comprising purified mRNA molecules. Optionally, the system may further include a filter for an additional filtration step (not shown). Said filter may include a filtration membrane with a pore size of less than 0.3 μm, for example 0.22 μm, for the final filtration step and thus can be used for the final filtration of a suspension comprising purified mRNA molecules obtained from the intermediate vessel.

The advantage lies in modification of the purified mRNA molecules, for example by dephosphorylation, by adding a 5'-cap or 3'-poly(A) tail. First of all, it is preferable to carry out steps (Ia)-(IIb) at least once before 3'-polyadenylation of the mRNA molecules, and steps (Ia)-(Ib) are preferably carried out at least once before 5'-capping. Optionally, the resulting mRNA molecules can be dephosphorylated after at least a first step (Ia) and at least a first step (Ib) followed by at least a second step (Ia) and at least a second step (Ib). Therefore, the system may further include one or more of the following structures (not shown): a second 'additional reservoir, preferably including the reagents required for capping, and preferably in fluid connection with the connecting hoses of the mRNA production system through a fourth' valve, a second 'additional reservoir' , preferably including the reagents necessary for dephosphorylation, and preferably in fluid connection with the connecting hoses of the mRNA production system through a fourth'' valve, and a second'' additional vessel, preferably including the reagents necessary for the enzymatic addition of the poly(A) tail, e.g. , at least poly(A) polymerase, and preferably in fluid connection with the connecting hoses of the mRNA production system through the fourth ''' valve. When the first valve and the intermediate valve are opened for a predetermined period of time, at least a portion of the respective suspension containing the mRNA molecules to be purified can be transferred from the TFF system to the intermediate vessel of the mRNA production system. When the first valve closes and the fourth', fourth' and/or fourth' gate opens, capping, dephosphorylation and/or polyadenylation of the mRNA molecules can be accomplished, preferably in the intermediate vessel. Alternatively, or additionally, if the reaction vessel is not a fermenter, the reaction vessel may be used in place of the intermediate vessel and thus open the second valve instead of the intermediate valve. When the fourth', fourth' and/or fourth' valve closes and the first valve opens, the suspension comprising the modified mRNA molecules can be transferred from the mRNA production system to the TFF system.

It should be noted that the filtration device may include more than one filtration membrane, which can be replaced automatically. Preferably, the filtration device includes more than one cell of the filtration device, for example, each including a filtration column, and the mRNA molecules are transferred for continuous filtration of the TFF into at least one of the cells containing a filtration membrane with a molecular weight cut-off as specified above for the relevant process step .

Moreover, as shown in FIG. 2, the system may include an outlet section, for example an outlet container in fluid communication with a reservoir, for example through a tenth valve. Preferably, said outlet container is in liquid connection or is in liquid connection with a sterile, preferably disposable, bag for storing at least a portion of the solution or suspension containing the purified mRNA molecules. When the tenth valve opens, purified mRNA molecules can be obtained. Alternatively, during the (last performed) step (IIb), the purified mRNA molecules may be contained in a filtrate rather than a concentrate. Optionally, the system may also include a filter for an additional filtration step (not shown). Said filter may comprise a filtration membrane with a pore size of less than 0.3 µm, for example 0.22 µm, for the final stage of filtration and thus can be used for the final filtration of a suspension containing purified mRNA molecules, for example a filtrate or suspension, obtained from the output tank. In addition, an auxiliary pump may be present to automatically control the rates of fluids transferred from the reservoir to the outlet section (not shown).

The system may further include preferably an automatically controlled cooling and/or heating element (not shown). Said cooling and/or heating element may be located at least substantially around one or more hose systems, containers (eg, reaction vessel, intermediate vessel, additional vessel and/or reservoir) and/or filtration device. Preferably, the cooling element is located at least partially around the reservoir and/or filtration device. Thus, the method of the present invention can be carried out at a temperature of from 0°C to 25°C, preferably for most stages from 2°C to 8°C, controlled using an appropriate cooling and/or heating element. In this way, large quantities of purified mRNA molecules can be obtained.

Preferably, the system may further comprise a preferably automatically adjustable pH control element (not shown). Said element may include a device for measuring the pH of the solution and/or suspension, such as a pH meter, and optionally a valve for regulating the pH, where the latter is preferably in fluid connection with at least one of the containers (for example, a reaction vessel, an intermediate vessel , additional capacity and/or reservoir). In this way, the pH of the solution and/or suspension in the appropriate container can be measured and/or controlled. Additionally or alternatively, the pH of the filtrate can be measured, preferably in a continuous manner. In this way, the mRNA purification process can be monitored with quality control.

In addition, the concentration and/or purity of the nucleic acid can be measured, preferably using the filtrate. This can be done photometrically by measuring the absorbance of molecules, including mRNA molecules, in the UV range at approximately 260 and 280 nm. For example, the ratio of the value obtained at about 260 nm to the value obtained at about 280 nm may indicate the purity of the corresponding nucleic acid in the solution or suspension. For example, a ratio of about 1.8 to about 2.0 may indicate high purity of the DNA or RNA molecules, while, for example, a ratio of less than 1.8 may indicate impurities, such as proteins. In this way, it is possible to obtain information about the composition of, for example, the filtrate, including the concentration of mRNA molecules. Optionally or alternatively, the concentration of the mRNA molecules can be determined based on, preferably, continuous conductivity measurements. Thus, the system may further preferably include an automatically controlled nucleic acid quantitation element (not shown), such as a spectrometer and/or spectrophotometer, for example a Nanodrop, and/or conductivity measuring devices.

Any or all of the containers (eg, reaction vessel, intermediate vessel, secondary chamber, and/or reservoir) may be positioned on appropriate scales. Thus, the mass of the corresponding container can be measured, the mass of the solution or suspension containing the mRNA molecules to be purified or purified in the corresponding container can be determined, and the corresponding amount of the corresponding solution or component added, for example, a first, second, third, fourth and/or fifth solution and/or or IVT reagents. In this way, the method for obtaining and/or purifying mRNA can be optimized and automated.

To provide automatic regulation of the method of obtaining and/or purifying mRNA, the system preferably includes controls designed to control the means described above, for example, valves, (auxiliary) pumps, heating and/or cooling element, pH control element, nucleic acid quantification element acids, means for carrying out conductivity and/or mass measurements. The term "system" as used in this context means a device and the terms may be used interchangeably.

With respect to the connecting hoses of the mRNA molecule production system, the hoses may be connecting hoses as shown in FIG. 2, for example, in liquid connection with a reaction vessel and other series-connected vessels. In another embodiment, the connecting hoses can be two or more hoses connecting the corresponding containers. Thus, the connecting hoses may, for example, mean at least a first hose system in fluid connection with the reaction vessel and the first additional vessel, and a second hose system in fluid connection with the reaction vessel and the second additional vessel. Preferably, one or more of said two or more hose systems comprises a valve between the respective containers and optionally an additional pump.

Any of the components of the system described above may be a disposable component. Preferably, one or more containers (eg, intermediate, reaction and/or additional container) and/or one or more (intermediate) valves may be corresponding disposable containers and valves, respectively. Preferably, the intermediate vessel and/or reaction vessel are disposable containers. Optionally, the entire system may be a single-use system, for example, for the production and purification of specific mRNA molecules. The advantage of such a system is that the corresponding mRNA molecule can be obtained and purified under sterile conditions, eliminating the time-consuming and costly washing, cleaning and/or sterilization steps that would otherwise be necessary to reduce the risk of contamination. Thus, preferably one or more components of the system described above are suitable disposable components.

The system, as shown by way of example in FIG. 2 has several advantages. In such a closed system, the risk of contamination such as RNase is minimized, while at the same time throughput can be increased since clogging of the filtration membranes can be avoided using continuous TFF filtration. Moreover, such a system can be used in a simple manner, for example using pump-controlled fluid transfer, and even in a fully automated manner, for example using automatically controlled (auxiliary) pumps and/or valves. Moreover, the system is easy to scale and customize to suit specific customer requirements. Thus, it is possible to carry out high-throughput purification of mRNA molecules in a highly efficient and automatic manner according to the method described herein using the system as shown in FIG. 2.

The present invention also relates to a method for preparing a pharmaceutical composition, comprising (a) purifying mRNA molecules according to the method for purifying mRNA molecules described above, and (b) processing the thus obtained mRNA molecules into a pharmaceutical composition. This is of particular interest for the pharmaceutical applications of purified mRNA molecules, such as the administration of mRNA molecules to promote the synthesis of a protein whose deficiency or defect is associated with a disease and/or the presence of which is required in a cell or has a beneficial effect on the cell.

Preferably, the function of the amino acid sequence that can be translated from the mRNA molecule purified according to the present invention in the cell or its environment is necessary or beneficial, for example, an amino acid sequence, the deficiency or defective form of which causes a disease or disorder, the provision of which can alleviate or prevent a disease or disorder, or an amino acid sequence that can accelerate a process beneficial to an organism, in a cell or in its environment. The encoded amino acid sequence may be a complete amino acid sequence or a functional variant thereof. In addition, the encoded amino acid sequence may act as a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where the amino acid sequence exhibits a function necessary to eliminate a disease , primarily metabolic disorders or to trigger in vivo processes, such as the formation of new blood vessels, tissues, etc. The term "functional variant" as used herein means a fragment that in a cell can exhibit the function of an amino acid sequence the function of which is essential in the cell or the deficiency or defective form of which is pathogenic.

Preferably, such an amino acid sequence is advantageous for use for complementary or medical purposes to create or restore physiological functions caused by the biosynthesis of suboptimal amino acid sequences, and thus also for direct or indirect beneficial effects on the course of diseases. Disorders with a known genetic basis include cystic fibrosis, hemophilia, hypertension, high cholesterol, cancer, neurodegenerative disorders, mental illness, etc. An online catalog with 22,993 records of genetic disorders and human gene disorders with corresponding genes and descriptions of their phenotypes is available at ONIM (Online Mendelian Inheritance in Man, http://onim.org) web page, all sequences are available in the Uniprot database (http://www.uniprot.org). As non-limiting examples in Table. 2 shows some congenital diseases and the corresponding gene(s). Due to the high level of interaction of cellular signaling pathways, mutations of a certain gene cause multiple pathogenic symptoms, of which only characteristic ones are given in Table. 2.

The protein can also cause an immunogenic reaction, for example as an antigen. Such proteins can be used for complementary or medical purposes, including vaccination.

In the case of the step of purifying the mRNA molecules, the same approaches are used as described above in connection with the method of purifying the mRNA of the present invention, wherein said method comprises steps (Ia) of purifying the precipitated mRNA molecules from a suspension comprising the precipitated mRNA molecules using a first solution, ( Ib) washing and dissolving the purified precipitated mRNA molecules obtained in step (Ia) using a second solution, (IIa) purifying the mRNA molecules from the dissolved mRNA molecules obtained in step (Ib) using a third solution including a chelating agent such EDTA, followed by step (IIb) of washing the purified mRNA molecules obtained in step (IIa) using a fourth solution, where steps (Ia) to (II) are performed using tangential flow filtration.

In the case of the step of processing the thus obtained purified mRNA molecules into a pharmaceutical composition, this step may include the addition of sodium chloride or citrate in the case of using water-purified mRNA molecules as the fourth solution in step (IIb) and in the last step (IIb) in the case of more than one stage (IIb), respectively. The advantage is that if the purified mRNA molecules were not obtained in a solution already containing sodium chloride or citrate, or if the purified mRNA molecules were obtained in a solution already containing sodium chloride or citrate, but at a concentration different from the concentration required by administered. In the latter case, the method may also include the step of determining the appropriate concentration and changing it if necessary. Preferably, the purified mRNA molecules are included in a solution that further contains sodium chloride or citrate at a concentration favorable for administration using a fourth solution comprising said sodium chloride or citrate at an appropriate concentration and a pH of 3 to 6, preferably 4 to 5, at (last) stage (IIb) of the method for purifying mRNA molecules described above.

Processing the resulting purified mRNA molecules into a pharmaceutical composition may include the step of adding a pharmaceutically acceptable carrier. The purified mRNA molecules are preferably included in an effective amount, i.e. in an amount sufficient to trigger a detectable therapeutic response in the subject to whom the pharmaceutical composition will be administered. The purified mRNA molecules and/or pharmaceutical composition may be present in sterile aqueous or non-aqueous solutions, suspensions and emulsions, as well as creams and suppositories, but may also be present in the form of powders, tablets or aerosols.

The term "pharmaceutically acceptable carrier" as used herein means chemical compounds, materials, ingredients and/or compositions that are medically acceptable for use in contact with human and animal tissues without excessive toxicity, irritation, or allergic reaction. or other problems or complications commensurate with an acceptable benefit/risk ratio. Thus, a pharmaceutically acceptable carrier is an inactive substance processed simultaneously with a pharmaceutically active substance to facilitate its use in terms of dose, adsorption, solubility or pharmacokinetic parameters.

Examples of suitable pharmaceutically acceptable carriers are well known in the art and include phosphate buffered saline solutions, buffering agents, water, emulsions such as oil/water emulsions, various types of wetting agents and sterile solutions. Primarily, aqueous vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffer media. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Other examples of pharmaceutically acceptable carriers include, but are not limited to, Ringer's solution and dextrose solution, citrate, phosphate and other organic acids, salt-forming counterions, e.g. sodium and potassium, low molecular weight (more than 10 amino acid residues) polypeptides, proteins, e.g. serum albumin or gelatin, hydrophilic polymers, e.g. polyvinylpyrrolidone, amino acids such as histidine, glutamine, lysine, asparagine, arginine or glycine, carbohydrates including glucose, mannose or dextrins, monosaccharides, disaccharides, other sugars such as sucrose, mannitol, trehalose or sorbitol, chelating agents such as EDTA, non-ionic surfactants such as polyoxyethylene sorbitan monolaurate, which are sold under the trade names Tween, propylene glycol, pluronics or polyethylene glycol, antioxidants including methionine, ascorbic acid and tocopherol, and/or preservatives e.g. octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens, e.g. methyl or propylparaben, catechol, resorcinol cyclohexanol, 3-pentanol and meta-cresol. Suitable pharmaceutically acceptable carriers and their compositions are described in more detail in the book Remington' Pharmaceutical Sciences, ^17th ed., (1985), Mack Publishing Co. In addition, preservatives, stabilizers and other additives may be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, nanosystems or liposomes, and the like.

The present invention also relates to a pharmaceutical composition comprising mRNA molecules purified by any appropriate methods described herein and obtained by the method described above. The pharmaceutical composition is advantageous in connection with regulating and/or enhancing translation of an amino acid sequence, such as a protein, in the cells of a subject to whom the pharmaceutical composition is administered, wherein the presence of said amino acid sequence, such as a protein, is beneficial and/or necessary for the subject, e.g. in the context of the disease.

With regard to the pharmaceutical composition of the mRNA molecules, their purification and preparation of the pharmaceutical composition, similar methods as described above can be used, including the features and advantages mentioned in the context of the corresponding embodiments of the present invention described above.

The pharmaceutical composition of the present invention can be administered using a variety of classes and methods of administration known to those skilled in the art, such as needle injection, inhalers, nebulizers, creams, foams, gels, lotions and ointments. The dosage and duration of action depend on the function performed by the purified mRNA molecules and must be specifically adjusted in each case. The duration of action should be as long as possible, for example, if these purified mRNA molecules are used for long-term treatment of a disease caused by a genetically deficient gene, while for other indications it can be regulated for the required time period. In addition, systemic administration of said purified mRNA molecules converted into suitable pharmaceutical compositions is possible.

Fig. 1. Schematic of a continuous tangential flow filtration (TFF) system with circulating mRNA molecules according to the method of the present invention. The mRNA molecules are in suspension in stage (Ia), dissolved in stage (Ib), and are in solution in the case of stages (IIa) and (IIb), respectively.

Fig. 2. Diagram of a typical system for purifying mRNA molecules according to the method of the present invention (with approximate locations of valves, (additional) pumps and/or (additional) containers).

Fig. 3. SDS-PAGE followed by colloidal Coomassie staining of the TFF concentrate, including unmodified tdTomato encoding mRNA molecules, after purification of the appropriate in vitro transcription mixture (IVT) in accordance with steps (Ia) and (Ib) at RT. Lane 1: enzyme mixture in ammonium acetate ( _NH4OAc ) as control, lane 2: enzyme mixture after filling the TFF system, lane 3: enzyme mixture after TFF in nuclease-free water, lane 4: IVT mixture, lane 5: mixture IVT after filling the system with TFF, lane 6: IVT mixture after TFF in nuclease-free water, lane 7: enzyme mixture in nuclease-free water as control. The enzyme mixture consists of RNase inhibitor, T7 RNA polymerase, inorganic pyrophosphatase and DNase I.

Fig. 4. SDS-PAGE followed by colloidal Coomassie staining of the TFF concentrate, including unmodified tdTomato encoding mRNA molecules, after purification of the corresponding in vitro transcription mixture (IVT) according to steps (Ia) and (Ib) at RT. Lane 1: enzyme mixture in nuclease-free water as control, lane 2: enzyme mixture before TFF, lane 3: IVT mixture after filling the TFF system, lane 4: IVT mixture after filtration in nuclease-free TFF water, lane 5: mixture IVT after TFF in nuclease-free water (after diafiltration in the presence of 2.5 M ammonium acetate and nuclease-free water, the corresponding mixture was concentrated 2.5 times to effectively detect protein removal). The enzyme mixture consists of RNase inhibitor, T7 RNA polymerase, inorganic pyrophosphatase and DNase I.

Fig. 5. SDS-PAGE followed by colloidal Coomassie staining of the TFF concentrate, including unmodified tdTomato encoding mRNA molecules, after purification of the corresponding in vitro transcription mixture (IVT) according to steps (Ia) and (Ib) at 4°C. Lane 1: Enzyme mixture in nuclease-free water as control, Lane 2: IVT mixture before TFF, Lane 3: IVT mixture after one volume wash (equal to original volume), Lane 4: IVT mixture after TFF in nuclease-free water, lane 5: IVT mixture after TFF in nuclease-free water (after diafiltration in the presence of 2.5 M ammonium acetate and nuclease-free water, the corresponding mixture was concentrated 2.5 times to effectively detect protein removal). The enzyme mixture consists of RNase inhibitor, T7 RNA polymerase, inorganic pyrophosphatase and DNase I.

Fig. 6. A) Western blot of hCFTR protein translated in HEK293 cells after transfection with hCFTR mRNA purified by TFF filtration and hCFTR mRNA purified by ammonium acetate precipitation followed by a 70% ethanol wash, reformulated using Lipofectamine Messenger Max. Hsp90 protein bands are shown as a control. Untransfected cells were used as negative controls. D) The amount of hCFTR expressed was assessed by densitometric analysis using Image Lab software and normalized to HSP90 protein. Untransfected cells (UT) were used as negative controls.

Fig. 7. Residual amounts of added nucleotides to non-hCFTR protein mRNA molecules compared to the amount of target mRNA molecules after varying percentage wash volumes. Filtration in tangential flow was carried out at CT.

Fig. 8. Residual amounts of added oligonucleotides to non-hCFTR mRNA molecules compared to the amount of target mRNA molecules after varying percentage wash volumes. Filtration in tangential flow was carried out at 4°C.

Fig. 9. Capillary gel electrophoresis using a Fragment Analyzer was performed using tdTomato mRNA, adding various DNA oligonucleotides (15 nt, 25 nt and 120 nt) containing an antisense DNA oligonucleotide (25 nt) before (A) and after (B) purification by TFF filtration at 4°C. The low molecular weight marker is an internal standard that is present in the Fragment Analyzer reagent kit to normalize the peak retention times in each cycle of capillary EP (15 nt RNA oligonucleotide).

Fig. 10. As an example, the filtrate volumetric flow rate was measured at various transmembrane pressures.

Fig. 11. Capillary gel electrophoresis using a Fragment Analyzer was performed using hCFTR mRNA with the addition of oligonucleotides of various DNA lengths (25 nt and 120 nt) and high molecular weight GLP-1 mRNA molecules (256 nt), before purification according to approach 1 ( A) and after stage (Ib) (B), after stage (IIa) (C), and after stage (IIb) (D) with CT. The small molecule marker is an internal standard that is present in the Fragment Analyzer reagent kit to normalize peak retention times in each cycle of capillary EP (15 nt RNA oligonucleotide).

Fig. 12. Capillary gel electrophoresis using a Fragment Analyzer was performed using hCFT mRNA with the addition of DNA oligonucleotides of various lengths (25 nt and 120 nt) and high molecular weight GLP-1 mRNA molecules (256 nt), before (A) and after (B) purification steps according to approach 2 at CT. The small molecule marker is an internal standard that is present in the Fragment Analyzer reagent kit to normalize peak retention times in each capillary EP cycle (15 nt RNA oligonucleotide).

Other objects and advantages of the present invention will be described in the following examples, which are intended to be illustrative but not limited to. All publications, patents, patent applications or other documents cited in this application are incorporated by reference in their entirety.

Examples

The methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are intended to be illustrative only, but not limited to.

The abbreviations used in this context and their corresponding descriptions are presented in Table. 3.

Materials, devices, software and analysis systems used

The materials are given in table. 4

The devices are listed in table. 5.

The software is shown in Table 6.

The system under study is shown in Table. 7.

Purification of an IVT mixture using TFF - stages (Ia) and (Ib)

An in vitro transcription (IVT) mixture comprising unmodified in vitro transcribed tdTomato targeting mRNA molecules containing 1644 nucleotides was used as described below. In addition, a mixture of enzymes was used, including T7 RNA polymerase, inorganic pyrophosphatase, RNase inhibitor and DNase I.

RNA precipitation

The IVT mixture and the enzyme mixture were each precipitated with an equal amount of ice-cold 5 M NH ₄ OAc at pH 7 to a final concentration of 2.5 M NH ₄ OAc and kept on ice for at least 30 minutes. Before TFF, the corresponding mixture was diluted 1:1 with 2.5 M NH ₄ OAc, pH 7, to a final concentration of mRNA of approximately 0.5 mg/ml in the mixture. The IVT mixture or enzyme mixture was passed through the TFF system, which was pre-washed for 10 min while circulating the IVT mixture at 50 ml/min with the filtrate valve closed.

Stage (Ia)

Removal of proteins, nucleotides and salts

Diafiltration of the corresponding mixture was carried out at a rate of 50 ml/min at a constant TMP of approximately 200-300 mbar using a 500 kDa MWCO mPES filtration column. The corresponding mixture was subjected to diafiltration with 10 volumes to wash with 2.5 M NH ₄ OAc pH 7. This step effectively removes enzymes, nucleotides, as well as buffer components contained in the mixture for in vitro transcription. Step (Ia) can be used to remove any other proteins and/or enzymes from any other intermediate production steps (eg dephosphorylation, post-capping, polyadenylation, ...). In the case of some enzymes (for example, poly(A) polymerase, which binds to the mRNA molecules of interest with high affinity), a surfactant (for example, SDS, LDS, ...) can be added to an ammonium acetate-based buffer solution with pH 7. In case poly(A) polymerase is used for polyadenylation, the subsequent step (Ia) is preferably carried out at a temperature in the range of 20°C to 30°C, preferably in the range of 23°C to 27°C, more preferably at 25°C WITH. This temperature is preferred for effective removal of poly(A) polymerase.

Stage (Ib)

Removal of NH ₄ OAc used in step (Ia)

A 100 kDa MWCO mPES diafiltration diafiltration column was used to remove NH ₄ OAc and to dissolve the mRNA molecules using 10 wash volumes using nuclease-free water at a flow rate of 50 ml/min and a TMP of approximately 200-300 mbar. Alternatively, depending on the size of the mRNA molecules, a 50 kDa MWCO mPES filtration column can be used.

To study the purification efficiency of steps (Ia) and (Ib) from a TFF concentrate containing mRNA molecules, samples can be taken before and after TFF (i.e. step (Ib)) and analyzed, for example, using a UV measurement analyzer for fragments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by, for example, staining with colloidal Coomassie dye.

As shown in FIG. 3-5, enzymes can be efficiently removed from mRNA molecules during steps (Ia) and (Ib) as described above. Thus, the mRNA molecules are retained in the TFF system while enzymes and/or proteins are efficiently removed. According to the diffuse peak analysis, the preliminary diffuse peak was 4.7% before TFF purification with a deviation of +1.3% for TFF purification at 4°C and +1.8% after TFF purification at RT. Thus, no degradation of mRNA was observed.

Purification of precipitated and dissolved mRNA using TFF Perform steps (IIa) and (IIb)

In the description below, precipitated and dissolved mRNA was used for further purification

Testing various filtration membranes and the effect of EDTA on mRNA transfer

Various filtration membranes with different pore sizes were tested for the transport of mRNA molecules using either nuclease-free water or nuclease-free water supplemented with 10 mM ethylenediaminetetraacetic acid (EDTA). Four mPES columns were tested with molecular weight cutoff (MWCO) values of 500 kDa, 300 kDa, 100 kDa, and 50 kDa, respectively. Transmembrane pressure (TMP) was set using the concentrate valve in the TFF system and maintained at 40 mbar for the 500 kDa and 50 kDa MWCO columns, respectively, and at 100 mbar for the 100 kDa and 300 kDa MWCO columns, respectively. The main pump speed was set to 15 ml/min and TFF was performed at RT. The initial concentration of mRNA molecules in the feed stream was 0.1 mg/ml, and the concentrations of mRNA molecules in the concentrate and filtrate were determined using UV measurements. Each column was tested by washing with 10 times the original sample volume.

In the absence of EDTA, the mRNA molecules are retained in the 500 kDa MWCO test column. It was assumed that the smaller pore sizes (i.e., 300 kDa, 100 kDa, and 50 kDa) did not allow passage of mRNA molecules and therefore further testing in the absence of EDTA was not performed.

In the presence of EDTA, mRNA molecules unexpectedly passed through the pores of the 500 kDa MWCO filtration membrane and also partially passed through the 300 kDa MWCO filtration membrane. However, mRNA molecules were successfully retained using 100 and 50 kDa MWCO filtration membranes in the presence of 10 mM EDTA. Therefore, the 100 kDa MWCO column was chosen to filter the mRNA molecules in the experiments presented below.

It should be noted that random precipitation of mRNA molecules was observed in some cases after filtration in the presence of 10 mM EDTA. However, such precipitation can be effectively prevented by using a buffer solution containing 40 mM MOPS and 10 mM EDTA.

Preparation of a buffer solution for diafiltration of 40 mM MOPS and 10 mM EDTA.

A buffer solution based on 1 M 3-(N-morpholino)propanesulfonic acid (MOPS) was prepared, where MOPS was dissolved in nuclease-free water. The pH of 1 M MOPS buffer solution was adjusted to 7 using 32% NaOH. The resulting 1 M MOPS pH 7 buffer solution was mixed with 0.5 M EDTA and nuclease-free water to produce a diafiltration buffer solution containing 40 mM MOPS and 10 mM EDTA.

Determination of wash buffer volumes to remove nucleic acid oligonucleotides

The efficiency of the TFF process, i.e. the minimum number of wash cycles required to completely remove contaminants such as nucleic acid oligonucleotides, which are immature transcripts and/or hydrolysis products, highly dependent on the repulsion of the molecule from the filter membrane and its pore size. Therefore, the corresponding effect of wash buffer volumes was examined to determine the number of wash cycles to remove added nucleic acid oligonucleotides from precipitated and dissolved mRNA including the mRNA molecules of interest.

For this experiment, the TFF flow rate was set to 15 mL/min and the TMP was kept constant at approximately 40 mbar using an mPES MWCO 100 kDa filtration column. The total feeding volume was 5 ml; DNA oligonucleotides 10 nucleotides long, constituting 2.5 wt., were added to 500 μl of mRNA molecules. % of the target mRNA molecule, 50 nucleotides, making up 2.5 wt. % of the target mRNA molecule and 120 nucleotides, making up 5 wt. % of the target mRNA molecule. Samples for reverse phase HPLC analysis were washed with 5, 10, 15, and 20 times the original volume of diafiltration feed buffer containing 40 mM MOPS and 10 mM EDTA. Before adding the sample, the filtrate valve and the concentrate valve were opened to circulate the concentrate at a rate of 50 ml/min for 5 min. For each wash cycle, 100 μL of sample was drawn through a sterile syringe and the samples were kept on ice until analyzed. TFF was performed at room temperature. Samples were examined using reverse phase HPLC analysis with peak areas detected at 260 nm corresponding to the relative amounts of DNA oligonucleotide in the sample. The corresponding areas before TFF (control) were taken as 100% oligonucleotide content.

In all cases, no nucleic acid oligonucleotides were detected after washing with 5-, 10-, 15-, and 20-fold wash volumes relative to the feed volume, respectively. Consequently, the results indicated that after 5 volumes of washing buffer, nucleic acid oligonucleotides in the concentrate were not detected. A minimum volume of 10 times the wash buffer was determined to be sufficient to remove nucleic acid nucleotides from mRNA molecules.

Determination of volumes for rinsing with nuclease-free water to remove EDTA

Since the removal of DNA oligonucleotides using TFF is carried out using a wash buffer solution, i.e. diafiltration buffer containing 40 mM MOPS and 10 mM EDTA, the subsequent filtration step is advantageous when replacing the wash buffer with nuclease-free water. Therefore, an experiment was performed to determine the number of wash cycles required to remove the wash buffer from the mRNA molecules. The feed solution from the previous experiment, containing the mRNA molecules and having a total volume of approximately 5 mL, which had previously been filtered with 20 times the volume of diafiltration wash buffer, was again diafiltered using nuclease-free water at a flow rate of 15 mL/min. at a constant TMP of 40 mbar using an mPES filtration column with a MWCO of 100 kDa. Samples for reverse phase HPLC analysis were taken after 5, 10, 15, and 20 times the initial feed volume was washed with nuclease-free water. To determine the amount of EDTA remaining after each wash cycle with nuclease-free water, a diafiltration buffer solution containing 40 mM MOPS and 10 mM EDTA was titrated and a standard calibration curve was recorded (see Table 3, MOPS-EDTA standard calibration curve: log(y )=0.6467 log(x)+l.9128, r=0.99462, r ² =0.99877, model curve: log/log). Since there is no absorption signal at 260 nm for the MOPS buffer solution, the concentrations given in Table. 8 and table. 9 correspond to EDTA concentrations. The experiment was carried out at RT 22°C. In table 9 data are shown after removing EDTA after each washing cycle.

As shown in table. 9, the reduced amounts of EDTA according to peak area determined by reverse phase HPLC after increasing wash volumes indicate that a minimum of 10 wash volumes of nuclease-free water are necessary to partially remove residual EDTA.

Testing specific parameters

Previous experiments conducted at RT determined the minimum number of wash cycles using diafiltration buffer required to remove DNA oligonucleotides during step (IIa), as well as the minimum number of wash cycles using nuclease-free water required to removal of residual EDTA during stage (IIb). To test certain parameters, nucleic acid oligonucleotides with a length of 10 nucleotides, corresponding to 2.5 wt., were added to 500 μg of mRNA. % target mRNA molecule, 50 nucleotides, corresponding to 2.5 wt. % of the target mRNA molecule and 120 nucleotides, corresponding to 5 wt. % volume of the target mRNA molecule in the total feed volume of 5 ml (called “before TFF” in Table 8 and Table 9).

For the experiments described below, the following conditions were used unless otherwise noted. The feed solution containing 0.1 mg/ml mRNA molecules was diafiltered using an mPES filtration column with MWCO 100 kDa at a flow rate of 15 ml/min and a TMP of approximately 40 mbar. The volumes used for flushing in both cases were 10x, i.e. 10 volumes of rinse with diafiltration buffer containing 40 mM MOPS and 10 mM EDTA, followed by a 10 volume wash of nuclease-free water (referred to as “post-TFF” in Table 8 and Table 9). Since mRNA molecules can decompose during hydrolysis, especially at elevated temperatures, experiments were carried out at 4°C, i.e. using ice water to ensure minimal hydrolysis of mRNA molecules.

These conditions resulted in efficient removal of DNA oligonucleotides of all three lengths tested (Table 10), as well as residual diafiltration buffer (Table 11), as demonstrated using reverse phase HPLC. In addition, the diffuse peaks from capillary gel electrophoresis were analyzed using a fragment analyzer before and after purification (Table 12). Analysis of the diffuse peaks indicates that the integrity of the mRNA was not altered during TFF purification and is therefore not affected by the process conditions.

Determination of translational efficiency of purified mRNA molecules

In addition, it was of significant interest to determine the translational efficiency of the resulting purified mRNA molecules. Therefore, 1.4 x 10 ⁶ HEK293 cells were seeded in 6-well plates and transfected with 3.75 μg of mRNA purified as described above and according to standard procedures, respectively. Transfection was performed using MessengerMax (1:15). After 24 h, the cells were lysed and examined by SDS-PAGE and Western blotting, respectively, using 50 mg of cell lysate in each case.

As can be seen in FIG. 6, a comparable number of mRNA molecules was detected using Western blotting (A) and quantified (B).

Determination of the threshold value for the removal of mRNA of different lengths

Various mRNA molecules of varying lengths were added to a total of 300 μg of hCFTR target mRNAs using a total volume of 5 ml. First, 6 different types of mRNA molecules were used to add each type corresponding to 16.7% based on the volume of target mRNA molecules. The three types of mRNA molecules contain a cap and a poly(A) tail, and are characterized by a total length of 3632 nucleotides, 1864 nucleotides and 1111 nucleotides, respectively. The two types contain neither a cap nor a poly(A) tail and are characterized by a total length of 494 nucleotides and 256 nucleotides, respectively. The latter type refers to a 120-nt DNA oligonucleotide that was used as a positive control.

The solution containing the added mRNA molecules was filtered as described above at RT (Figure 7) and at 4°C (Figure 8), respectively. Samples were washed prior to TFF (immediately upon sample receipt) with 5, 10, 15, and 20 wash volumes per initial sample volume using diafiltration buffer as described above, and after an additional wash using 10 nuclease-free wash volumes water. For each wash cycle, 100 μL of sample was washed using a sterile syringe and kept on ice until analysis. Passage through the filter pores was assessed using capillary gel electrophoresis using a fragment analyzer.

As shown in FIG. 7 and Fig. 8, the cutoff for mRNA molecules of at least 951 nucleotides in length is observed using a 100 kDatPE8 column. For mRNA molecules less than 951 nucleotides in length, lower MWCO columns are preferred, such as 50 or 70 kDa mPES MWCO columns.

Increasing the concentration of mRNA molecules from 0.1 mg/ml to 1 mg/ml

In the final set of experiments below (Figure 9), the filtration procedure described below was applied to a solution containing the unmodified 1644 nucleotide tdTomato mRNA as the target mRNA molecule. First of all, we analyzed the total volume of the feeding solution, including 1 mg/ml of mRNA molecules, corresponding to a total amount of 5 mg of mRNA molecules, as well as a DNA oligonucleotide with a length of 15 nucleotides and an antisense oligonucleotide with a length of 25 nucleotides, which complementarily binds to the studied mRNA molecules, and represents 2. 5 wt. % of the target mRNA molecule, and oligonucleotides 120 nucleotides long, representing 5 wt. % of the target mRNA molecule. This added feed solution was diafiltered at 4° C. using an mPES MWCO 100 kDa filtration column at a flow rate of 15 ml/min and a TMP of approximately 40 mbar. The wash volumes used were i) 10 volumes of nuclease-free water, followed by ii) 10 volumes of diafiltration buffer containing 40 mM MOPS and 10 mM EDTA, followed by iii) 10 volumes of nuclease-free water.

The magnitude of the diffuse peaks was examined as an indicator of mRNA integrity. First, the following blurred peak values were obtained: a pre-blurred peak of 6.4% before TFF and a pre-blurred peak of 5.9% after TFF. The areas of the preliminary diffuse peaks correspond to the proportion of mRNA in relation to hydrolyzed products. Thus, there are no hydrolysis products based on fragment analysis, and it has been established that TFF purification does not affect the integrity of the mRNA.

Typical scheme for determining optimal TFF parameters using standard measurements

The following is a brief description of the optimization of the TFF method for those skilled in the art as described above using standard measurements

Stage (Ia)

The TFF flow rate can be adjusted to provide sufficient shear to recycle the precipitated mRNA molecules within the TFF system and to wash the precipitated mRNA molecules off the filter surface. Sufficient filtrate flow is ensured by opening the filtrate valve. Enzyme removal can be determined, for example, by SDS-PAGE and the required wash volumes can be adjusted by standard measurements (preferably 1-20 wash volumes, more preferably 5-15 wash volumes, more preferably 9-11 wash volumes). One rinse volume can be defined as a volume equal to the volume of diafiltration medium, such as ammonium acetate buffer pH 7, based on the initial feed volume.

Stage (Ib)

Similar parameters can be used as described in step (Ia). In this case, the buffer solution can be replaced, for example, with nuclease-free water to dissolve the mRNA molecules and to remove the buffer substance (for example, ammonium acetate). Buffer removal can be determined, for example, by conductivity measurements, UV absorbance measurements, and required wash volumes can be determined by standard measurements (preferably 1-20 volumes, more preferably 5-15 volumes, more preferably 9-11 wash volumes). One rinse volume can be defined as a volume equal to the volume of diafiltration medium, such as ammonium acetate buffer pH 7, based on the initial feed volume.

Stage (IIa)

The TFF flow rate can be adjusted to provide sufficient shear to recycle the precipitated mRNA molecules within the TFF system and to wash the precipitated mRNA molecules off the filter surface. Sufficient filtrate flow is ensured by opening the filtrate valve. Loss of the target mRNA can be monitored, for example, by measuring UV absorbance (eg, system off or on) in the filtrate line. If loss of the target mRNA is observed, the flow rate and/or TMP can be adjusted by standard measurements until no further loss of the target mRNA is observed. The required wash volumes can be adjusted by standard measurements depending on the amount of impurities (eg, immature transcripts and/or hydrolysis products) (preferably 1-20 volumes, more preferably 5-15 volumes, more preferably 9-11 wash volumes). One rinse volume can be defined as a volume equal to the volume of diafiltration medium, such as ammonium acetate buffer pH 7, based on the initial feed volume.

Stage (IIb)

Similar parameters can be used as described in step (IIa). In this case, the buffer solution can be replaced, for example, with nuclease-free water to remove the buffer solution, for example, MOPS-EDTA. Buffer solution removal can be determined, for example, by conductivity measurement, UV absorbance measurement, and required wash volumes can be determined by standard measurements (preferably 1-20 volumes, more preferably 5-15 volumes, more preferably 9-11 wash volumes). One rinse volume can be defined as a volume equal to the volume of diafiltration medium, such as ammonium acetate buffer pH 7, based on the initial feed volume.

Experiments to control TMP can help determine optimal conditions for the purification of mRNA molecules. For example, a total feed solution volume of 5 mL, including a concentration of target mRNA molecules of 1.0 mg/mL in the diafiltration buffer solution used in step (IIa) as described above, was diafiltered using a 100 kDa mPES MWCO filtration column and flow rate 15 ml/min. The feed flow was measured in ml/min at 6 different TMP values ranging from approximately 50 mbar to approximately 150 mbar. As can be seen in FIG. 10 for tdTomato mRNA, the optimal TMP is approximately 50 mbar at a flow rate of 15 ml/min, but may depend on the experimental conditions.

Comparison example

Below is an example performed to compare the results obtained by the method described in this context (approach 1) and the method described in the application WO 2015/164773 A1 (approach 2).

Approach 1

In vitro transcribed unmodified hCFTR mRNA (see US patent application 9713626 B2, 5 mg pellet) was supplemented with 25 nucleotide (nt) and 120 nucleotide (nt) DNA oligonucleotides, as well as 256 nucleotide glucagon-like peptide 1 mRNA molecules (GLP-1 mRNA molecules (SEQ ID NO: 1). DNA oligonucleotides 25 nucleotides long were added to mRNA in an amount of 2.5% based on the total amount of mRNA in μg. DNA oligonucleotides 120 nucleotides long were introduced into mRNA in an amount of 5.0 % based on the total amount of mRNA in μg. GLP-1 mRNA molecules with a length of 256 nucleotides were added to the mRNA in an amount of 16% based on the total amount of mRNA in μg.

The added mRNA molecules were precipitated using 2.5 M NH ₄ OAc (pH 7).

To remove proteins, salts and immature transcripts, step (Ia) was carried out using 10 wash volumes with 2.5 M NH ₄ OAc and an mPES MWCO 500 kDa filtration column, flow rate 15 ml/min and TMP 200-300 mbar (mRNA concentration: 0.5 mg/ml).

To remove NH ₄ OAc and to dissolve the mRNA molecules, step (Ib) was carried out using 10 wash volumes of nuclease-free water and an mPES MWCO 50 kDa filtration column, flow rate 15 ml/min and TMP 200-300 mbar (mRNA concentration: 0 .5 mg/ml).

To remove divalent cations, immature transcripts and/or hydrolysis products, step (IIa) was performed using 10 wash volumes of 40 mM MOPS and 10 mM EDTA and an mPES filtration column with MWCO 100 kDa, flow rate 15 ml/min and TMP approximately 20 mbar (mRNA concentration: 1.0 mg/ml).

To remove the MOPS-EDTA diafiltration buffer, step (IIb) was performed using 10 wash volumes of nuclease-free water and a 100 kDa mPES MWCO filtration column, flow rate 15 ml/min and TMP approximately 20 mbar (mRNA concentration: 1.0 mg/ml).

The resulting mRNA molecules were examined using a fragment analyzer/reverse phase HPLC to monitor the removal of added oligonucleotides and a Nanodrop instrument to determine the recovery of the mRNA molecules.

Approach 2 (described in WO 2015/164773 A1)

In vitro transcribed unmodified hCFTR mRNA (see US patent application 9713626 B2, 10.26 mg pellet) was supplemented with DNA oligonucleotides of 25 nucleotides (nt) and 120 nucleotides (nt), as well as glucagon-like peptide 1 mRNA molecules 256 nucleotides long (GLP-1 mRNA molecules (SEQ ID NO: 1). DNA oligonucleotides 25 nucleotides (nt) long were added to the mRNA in an amount of 2.5% based on the total amount of mRNA in μg. DNA oligonucleotides 120 nucleotides long were added to mRNA in an amount of 5.0% based on the total amount of mRNA in μg. GLP-1 mRNA molecules with a length of 256 nucleotides were added to the mRNA in an amount of 16% based on the total amount of mRNA in μg.

The added mRNA molecules were precipitated using i) guanidinium thiocyanate, sodium lauryl sarcosyl and sodium citrate to final concentrations of 2.09 M guanidinium thiocyanate, 0.26% sodium lauryl sarcosyl and 13.0 mM sodium citrate and ii) absolute ethanol to a final concentration of ~38% CN. and incubated for 5 min at RT.

Application was carried out using approximately 22 ml of precipitated mRNA at a flow rate of approximately 6 ml/min using an mPES MWCO 500 kDa filtration column (mRNA concentration: 0.52 mg/ml).

The washing was carried out by repeating the following two steps more than 5 times at a flow rate of approximately 6 ml/min using an mPES MWCO 500 kDa filtration column (mRNA concentration: 0.52 mg/ml): step a) washing with 5 ml of 2.09 M guanidinium thiocyanate, 0.26% sodium lauryl sarcosyl and 13.0 mM sodium citrate, ~38% EtOH, and step b) wash with 5 ml 80% ethanol.

Elution (step B) was accomplished by treating the resulting solid mRNA with 5 ml of nuclease-free water and recirculating for 5-10 min (with the filtrate valve closed) to ensure dissolution. This procedure was repeated until mRNA molecules were detected using a flow rate of approximately 6 ml/min using an mPES MWCO 500 kDa filtration column (mRNA concentration: 0.52 mg/ml).

Dialysis (step D) was performed using approximately 5 wash volumes containing 1 mM sodium citrate (pH 6.4), a flow rate of approximately 6 ml/min and a 500 kDa mPES MWCO filtration column (mRNA concentration: 0.52 mg/ml).

The resulting mRNA molecules were examined using a fragment analyzer/reverse phase HPLC to remove oligonucleotide impurities and a Nanodrop instrument to determine the yield of the resulting mRNA molecules.

It should be noted that the following changes were used in the case of approach 2 described above, compared to the method described in WO 2015/164773 A1: After linear downscaling to maintain a constant shear rate in the filtration column, the initially planned flow rate was 6 ml /min. However, this flow rate resulted in a very low TMP, i.e. the effective filtrate flow rate was 0 ml/min and diafiltration was not possible. Therefore, the flow rate was increased stepwise until sufficient TMP was recorded, resulting in a significant filtrate flow. Thus, the final flow rate used in steps a) - d) was in the range of 20-24 ml/min (filtrate flow rate from 1.1 to 1.25 ml/min).

The results shown in table. 13 were obtained using a fragment analyzer, which was performed on crude and purified hCFTR mRNA samples. The corresponding electropherograms are presented in Fig. 11 for approach 1 and FIG. 12 for approach 2, respectively.

In the case of approach 1, removal of oligonucleotide impurities (25 nucleotides and 120 nucleotides) was mainly achieved in step (Ia)/(Ib). Stage (IIa) is necessary to remove 256 nucleotides long mRNA, which models long hydrolysis products. In step (IIb), no additional removal of the 256 nucleotide long mRNA occurs, but in turn, removal of hydrolysis products and immature sequences occurs (step (IIa) using a highly effective chelating agent such as EDTA.

In the case of approach 2, removal of oligonucleotide impurities (25 nucleotides and 120 nucleotides) was observed only partially at stages A - B. At stage D, DNA oligonucleotides 120 nucleotides long were not completely removed. mRNA of 256 nucleotides in length, simulating long hydrolysis products, remained almost completely in the sample after TFF purification. Thus, dialysis with 1 mM sodium citrate (step D) does not have a significant effect on the additional removal of 120 nucleotide DNA oligonucleotides, which model long hydrolysis products.

The 256 nucleotide mRNA sequence used in this context is described in detail below.

SEQ ID No: 1

Codon-optimized GLP-1 sequence

part of the T7 promoter, C: etris minimal 5'UTR followed by an additional nucleotide U, TISU 5'UTR, start codon, mRNA codon-optimized GLP-1 sequence, stop codon. part of an EcoRI restriction site.

For experiments with the addition of oligonucleotides, non-polyadenylated mRNA was used.

Claims (22)

1. A method for purifying mRNA molecules, where said method includes (Ia) purifying the precipitated mRNA molecules from the suspension comprising the precipitated mRNA molecules using the first solution, (Ib) washing and dissolving the purified precipitated mRNA molecules obtained in step (Ia) using a second solution, (IIa) purifying the mRNA molecules from the dissolved mRNA molecules obtained in step (Ib) using a third solution containing a chelating agent, and then (IIb) washing the purified mRNA molecules obtained in step (IIa) using the fourth solution, where stages (Ia)-(IIb) are carried out using tangential flow filtration. 2. The method according to claim 1, where the chelating agent is EDTA. 3. Method according to claim 1 or 2, where the pH value of the third solution is in the range from 1 to 10. 4. The method of any one of the preceding claims, wherein the third solution comprises a MOPS buffer. 5. The method according to any of the previous paragraphs, where stages (Ia)-(IIb) are carried out at a temperature from 0°C to 25°C. 6. The method as claimed in any one of the preceding claims, wherein before step (Ia), said suspension is prepared using ammonium acetate to precipitate the mRNA molecules, and wherein the first solution contains ammonium acetate. 7. A method according to any of the preceding claims, wherein the second solution is water or where the fourth solution is water or includes sodium chloride and/or citrate. 8. The method according to any of the preceding claims, wherein the mRNA molecules are contained in the concentrate after filtration in a tangential flow. 9. The method according to claim 8, where the concentrate obtained at stage (Ia) is used as a feed stream of the solution for filtration in tangential flow at stage (Ib), the concentrate obtained at stage (Ib) is used as a feed stream of the solution in step (IIa), and the concentrate obtained in step (IIa) is used as a feed solution in step (IIb). 10. The method according to any of the preceding claims, wherein the mRNA molecules contained in the suspension are obtained by in vitro transcription. 11. The method according to any of the preceding claims, where the method further comprises dephosphorylation and/or polyadenylation and/or post-capping of mRNA molecules. 12. The method according to claim 11, where the method includes dephosphorylation of the mRNA molecules obtained at stage (Ib), followed by stages (Ia)-(IIb), followed by polyadenylation of the resulting mRNA molecules, followed by stages (Ia)-( IIb). 13. The method according to any of the preceding paragraphs, where in the case of stage (Ia) a filtration membrane with a molecular weight cut-off limit of 300 kDa to 0.65 μm is used for filtration in tangential flow, and/or where in the case of stages (Ib) and (IIb) use a filtration membrane with a molecular weight cut-off limit of 1 kDa to 0.65 μm, and/or in the case of step (IIa) use a filtration membrane with a molecular weight cut-off limit of at least 70 kDa. 14. The method according to any of the preceding claims, wherein the volume of diafiltration of any first, second, third and/or fourth solution is 1 times the volume of the suspension in stage (Ia). 15. A method for producing a pharmaceutical composition, including (a) purifying mRNA molecules by the method according to any one of claims. 1-14 and (b) processing the thus obtained mRNA molecules into a pharmaceutical composition.

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